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UniversityM icrofilms

International300 N. Zeeb Road Ann Arbor. Ml 48106

8400241

Kuzm anoff, Konrad Marc

INVESTIGATIONS INTO THE INVOLVEMENT OF CALCIUM AND CALMODULIN IN THE MEDIATION OF MAIZE ROOT GROWTH

The Ohio S ta te University Ph.D.

University Microfilms

International 300 N. Zeeb Road, Ann Arbor, Ml 48106

INVESTIGATIONS INTO THE INVOLVEMENT OF CALCIUM AND CALMODULIN

IN THE MEDIATION OF MAIZE ROOT GROWTH

DISSERTATION

P r e s e n t e d i n P a r t i a l F u l f i l l m e n t o f t h e R e q u i r e m e n t s f o r

t h e Degree D oc to r o f P h i l o s o p h y i n t h e G r a d u a te

S choo l o f The Ohio S t a t e U n i v e r s i t y

By

Konrad Marc Kuzmanoff , B . A . , M.Sc.

* * * * *

The Ohio S t a t e U n i v e r s i t y

1983

R ead ing Commit te : Approved By

M ic ha e l L. Evans

George A. B a r b e r ~22lA d v i s e r

G a ry L. F l o y d M o l e c u l a r , C e l l u l a r andD e v e l o p m e n ta l B i o l o g y

Roy A. T a s s a v a

DEDICATION

D e d i c a t e d t o a modern f a r m e r , my g r a n d f a t h e r ,

J u l i u s Edward P l i n e .

i i

ACKNOWLEDGMENTS

I wish to thank my parents for their belief in my abilities,

without whom this dissertation would not have been possible.

I w i s h t o acknow ledge my a d v i s o r , Dr. M ic h a e l L. E v a n s , f o r h i s

a d v i c e i n t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t and d u r i n g my

d i s s e r t a t i o n r e s e a r c h p r o j e c t . I w i s h t o t h a n k Dr. Evans f o r h i s

e n t h u s i a s m and b e l i e f i n my a b i l i t i e s t o c o m p l e t e t h i s p r o j e c t . I

have e n j o y e d b e i n g a b l e t o work w i t h and l e a r n from him.

I also wish to extend my gratitude to the members of my reading

committee for their advice and valued opinions during the course of

this research.

I wish to thank Jody Banks for many rewarding discussions and

much appreciated support during this research.

I a l s o w i s h t o t h a n k Alpha B i t Com munica t ions f o r w r i t i n g t h e

word p r o c e s s i n g p ro g ra m ' L a z y w r i t e r ' and t o t h a n k Tandy C o r p o r a t i o n

f o r d e s i g n i n g and m a r k e t i n g t h e TRS-80 model I and model I I I

m i c r o c o m p u t e r s , w i t h o u t which t h i s d i s s e r t a t i o n may n e v e r have b e e n

w r i t t e n .

VITA

1974 ............................................ B . A . , The J o h n s Hopkins U n i v e r s i t y ,B a l t i m o r e , Md., o r g a n i c c h e m i s t r y .

1974-1976 ................................ R e s e a r c h A s s o c i a t e , D e p t . B i o l o g y , ColumbiaU n i v e r s i t y , New York.

1976-1979 ................................ R e s e a r c h and T e a c h i n g A s s o c i a t e , D ep t , o fB o t a n y , The Oh io S t a t e U n i v e r s i t y , Columbus, OH.

1979 . . . . . . . . . . . . . . . . . . . M . S c . , The D e p t , o f B o t a n y ,The Ohio S t a t e U n i v e r s i t y , Columbus, OH.

1979-1982 ................................ R e s e a r c h and T e a c h i n g A s s o c i a t e ,M o l e c u l a r , C e l l u l a r and D e v e lo p m e n ta l B i o l o g y P r o g ra m , The Ohio S t a t e U n i v e r s i t y , Columbus, OH.

1982-1983 ................................ U n i v e r s i t y P r e s i d e n t i a l F e l l o w s h i p ,The Oh io S t a t e U n i v e r s i t y , Columbus, OH.

P u b l i c a t i o n s

Kuzmanoff K.M. 1981. S o r t M o d i f i c a t i o n . TRS-80 M ic rocom pu te r News ^ : 5 - 6

Kuzmanoff K.M. and M.L. Evans 1978. A d a p t a t i o n i n t h e g row th o fL e n t i l r o o t s t o s u d d e n t u r g o r s h i f t s . Oh io J . S c i . 78 :17

Kuzmanoff K.M. and M.L. Evans 1978. E f f e c t s o f o s m o t i c a l l y - i n d u c e dt u r g o r s h i f t s on g r o w th and t h e a c i d r e s p o n s e i n l e n t i l r o o t s . P l a n t P h y s i o l . 61^:43, S u p p l .

Kuzmanoff K.M. and M.L. Evans 1979. Rap id a d a p t a t i o n o f r o o t g row tht o o s m o t i c s t r e s s . P l a n t P h y s i o l . j63 :140 , S u p p l .

Kuzmanoff K.M. and M.L. Evans 1980. C h a r a c t e r i z a t i o n o fa c i d - e n h a n c e m e n t o f w a l l l o o s e n i n g i n r o o t s o f s oybe an and c o r n . P l a n t P h y s i o l . 6>5:130, S u p p l .

Kuzmanoff K.M. and M.L. Evans 1981. D e t e r m i n a t i o n o f t h e f r e e spa cep r e s e n t i n p r i m a r y r o o t s o f l e n t i l . Oh io J . S c i . 81 :35

Kuzmanoff K.M. and M.L. Evans 1981. D i g i t z e d m easu rem en ts o f s h o r t t e r m g rowth k i n e t i c s o f s o y b e a n , c o r n and l e n t i l r o o t s . Oh io J . S c i . 8 1 :36

Kuzmanoff K.M. and M.L. Evans 1981. K i n e t i c s o f a d a p t a t i o n t oo s m o t i c s t r e s s i n l e n t i l (Lens c u l i n a r i s Med.) r o o t s . P I .P h y s i o l . 6 8 : 2 4 4 -2 4 7

Kuzmanoff K.M. and M.L. Evans 1981. C h a r a c t e r i z a t i o n o f a componenta p p a r e n t l y i n v o l v e d i n t h e a c i d - i n d u c e d g row th r e s p o n s e o f r o o t s .Oh io J . S c i . 8 1 :3 6

Kuzmanoff K.M. and M.L. Evans 1982. A c a l m o d u l i n - l i k e p r o t e i n i nc o r n r o o t s : E v i d e n c e f o r i t s e x i s t e n c e and p o t e n t i a l i n v o lv e m e n ti n r o o t g r o w th . P I . P h y s i o l . ( j9 :316, S u p p l .

Kuzmanoff K.M. and R .S . P l a t t 1978. An i n s t r u m e n t t o i n t e g r a t er a d i a n t e n e r g y d o s a g e i n f i e l d m ea su rem e n ts o f p h o t o s y n t h e s i s .O h io J . S c i . 7 8 :20

M a n c i n e l l i A . L . , Yang C -P H . , R a b in o I . and K.M. Kuzmanoff 1976. P h o t o c o n t r o l o f a n t h o c y a n i n s y n t h e s i s V. F u r t h e r e v i d e n c e a g a i n s t t h e i n v o lv e m e n t o f p h o t o s y n t h e s i s i n h i g h i r r a d i a n c e r e a c t i o n s y n t h e s i s o f young s e e d l i n g s . P I . P h y s i o l . 5 8 : 2 1 4 -2 1 7

Mulkey T . J . , Evans M . L . , and K.M. Kuzmanoff 1983. The k i n e t i c s o f a b s c i s i c a c i d a c t i o n on r o o t g ro w th and g r a v i t r o p i s m . P l a n t a 1 5 7 : 1 5 0 - 1 5 7 .

Mulkey T . J . , Kuzmanoff K.M. and M.L. Evans 1981. C o r r e l a t i o n b e tw e en p r o t o n - e f f l u x p a t t e r n s and g ro w th p a t t e r n s d u r i n g g e o t r o p i s m and p h o t o t r o p i s m i n m a iz e and s u n f l o w e r . P l a n t a 152:239-241

Mulkey T . J . , Kuzmanoff K.M. and M.L. Evans 1982. P r o m o t io n o f g row th a nd h y d ro g en io n e f f l u x by a u x i n i n r o o t s o f m aize p r e t r e a t e d w i t h e t h y l e n e b i o s y n t h e s i s i n h i b i t o r s . P l a n t P h y s i o l o g y 7 0 : 1 8 6 - 1 8 8 .

Rab ino I . , M a n c i n e l l i A.L. and K.M. Kuzmanoff 1977. P h o t o c o n t r o l o f a n t h o c y a n i n s y n t h e s i s VI. S p e c t r a l s e n s i t i v i t y , i r r a d i a n c e d e p e n d e n c e , and r e c i p r o c i t y r e l a t i o n s h i p s . P l a n t P h y s i o l . 5 9 : 5 6 9 - 5 7 3

v

TABLE OF CONTENTS

DEDICATION ................................................................................................................................. i i

ACKNOWLEDGMENTS ..................................................................................................................... i i i

V I T A ................................................................................................................................................ iv

LIST OF TABLES .................................................................................................................... v i i i

LIST OF FIGURES .................................................................................................................... i x

LIST OF PLATES ....................................................................................................................... x i

LIST OF ABBREVIATIONS ...................................................................................................... x i i

INTRODUCTION ............................................................................................................................ 1

C h a p t e r

I . MEASUREMENT OF THE PLASTIC AND ELASTIC COMPLIANCE OF MAIZE ROOT TISSUE AND THE INDUCTION OF CELL WALL LOOSENING BY ACID AND EDTA ....................................................................... 16

I n t r o d u c t i o n .................................................................................................... 16

M a t e r i a l s and Methods .............................................................................. 19

R e s u l t s and D i s c u s s i o n ............................................................................ 23

I I . MEASUREMENT OF ROOT GROWTH AS AFFECTED BY CALCIUM ANDINHIBITORS OF CALMODULIN ............................................................................ 52

I n t r o d u c t i o n ................... 52



I I I . EXAMINATION OF THE PROTEINACEOUS MATERIAL RELEASED FROMMAIZE ROOTS BY OSMOTIC SHOCK.................................................................. 93

I n t r o d u c t i o n .................................................................................................... 93


R e s u l t s and D i s c u s s i o n ............................................................................ 97v i

IV. QUANTIFICATION OF EXTRACTABLE CALMODULIN FROM ROOTSOF MAIZE AND LEAVES OF SPINACH ............................................................. 133

I n t r o d u c t i o n .................................................................................................... 133



SUMMARY ......................................................................................................................................... 172

LIST OF REFERENCES .............................................................................................................. 176

APPENDIXES

A. V e r t i c a l S l a b Gel P r o c e d u r e s ................................................................... 187

B. P r e p a r a t i o n o f Gel S o l u t i o n s ................................................................... 194

C. S t a i n i n g o f P o l y a c r y l a m i d e Gels ............................................................ 209

D. SDS P o l y a c r y l a m i d e G r a d i e n t Gels .......................................................... 222

E. P h o s p h o d i e s t e r a s e A ssay f o r C a lm o d u l in ........................................... 225

F. A ssa y f o r ATPase A c t i v i t y .......................................................................... 229

G. R ad io - im m unoassay (RIA) f o r C a lm o d u l in ........................................... 230

H. Listing of Data Aquisition Program, AQUIRE/BAS,Used t o M easure Root G r o w t h ..................................................................... 233

I. Listing of Data Manipulation Program, CHANGE/BAS,Used t o A d j u s t D i g i t i z e d D a ta ................................................................ 248

J. Listing of Linear Regression Program, LINREG/BAS,Used t o C a l c u l a t e B e s t F i t S t r a i g h t , L o g a r i t h m i c , and E x p o n e n t i a l Curves ........................................................................ 255

K. Listing of Polynomial Curve Fitting Program,MATRIX2/BAS ............................................................................................................ 259

v i i

LIST OF TABLES

T a b l e

1. C a l c u l a t e d E l a s t i c and P l a s t i c F r a c t i o n s o f S t r e s s -I n d u c e d E x t e n s i o n s o f Maize Roo t s .................................................... 34

2. Com pari son o f t h e E f f e c t o f C a lc iu m C o n c e n t r a t i o n Upont h e I n h i b i t i o n and R e c o v e ry o f t h e Growth R a t e o f Maize R o o t s ....................................................................................................................... 63

3. Compari son o f C a l c u l a t e d Growth R a t e B e f o r e and A f t e rA d d i t i o n o f C a l c i u m ...................................................................................... 65

4. I n h i b i t i o n o f Maize Roo t Growth by C h lo r p ro m a z i n e .............. 83

5. OSM Sephadex G-100 P eaks and T h e i r C a l c u l a t e dM o l e c u l a r W eigh t s .......................................................................................... I l l

6 . OSM Sephadex G-50 P eaks and T h e i r C a l c u l a t e dM o l e c u l a r W eigh t s .......................................................................................... 115

7. Compari son o f F i t t e d C urves Used t o C a l c u l a t e CaMC o n t e n t o f OSM Us ing R ad io - Im m unoassay ....................................... 127

8. P r e l i m i n a r y Measurement o f ATPase A c t i v i t y o f t h e OSM . . 130

9. Com pari son o f S p e c i f i c and T o t a l C a lm o d u l in A c t i v i t y D u r in g P r o g r e s s i v e P u r i f i c a t i o n from Maize R oo t sand S p in a c h Leaves ........................................................................................ 151

29

32

36

39

41

44

46

48

61

68

71

73

75

LIST Of FIGURES

Measurement o f E l a s t i c and P l a s t i c Components o f I n t a c t F rozen /T haw ed Maize R oo t s ......................................................

D e t e r m i n a t i o n o f t h e Y i e l d S t r e s s o f I n t a c t ,F / T Maize R oo t s ................................................................................................

A c i d - and EDTA-Induced I n c r e a s e i n Maize Root E x t e n s i b i l i t y ....................................................................................................

E s t i m a t i o n o f P l a s t i c and E l a s t i c ComponentsA f t e r T r e a t m e n t o f F /T T i s s u e w i t h EDTA .....................................

E f f e c t o f EDTA Upon A c i d - I n d u c e d E x t e n s i o n o fF / T Maize R oo t s ................................................................................................

E f f e c t o f I n c r e a s i n g C o n c e n t r a t i o n s o f EDTA UponC e l l Wall L o o s e n in g o f F roz en /T ha w e d Maize R o o t s ...............

EDTA Mimicry o f A c i d - I n d u c e d Growth i n L i v i n g ,I n t a c t Maize Roo t s ........................................................................................

L oss o f A c i d - I n d u c e d E x t e n s i o n A f t e r T r e a t m e n tw i t h Urea ..............................................................................................................

Compari son o f t h e E f f e c t o f I n c r e a s i n g C o n c e n t r a t i o n s o f C a lc ium Upon t h e Growth o f Maize S e e d l i n g R o o t s ..........

R a p id O s c i l l a t i o n s i n t h e C a l c i u m - I n d u c e d I n h i b i t i o n o f Root Growth ..................................................................................................

Compari son o f t h e E f f e c t o f C a l c iu m Upon t h e C a l c u l a t e d ' I n s t a n t a n e o u s ' Growth R a t e o f Maize R o o t s ..............................

E f f e c t o f 50 mM M a n n i t o l Upon Root Growth ................................

Com pari son o f I n h i b i t i o n o f Growth by M a n n i t o l and C a l c i u m ...................................................................................................................

ix

14. Ac id S t i m u l a t i o n o f Root G r o w t h . i n t h e P r e s e n c eo f C a l c i u m ............................................................................................................ 80

15. Inhibition of Root Growth by Calmodulin Inhibitors .......... 85

16. I n h b i t i o n o f Root Growth by C h l o r p r o m a z i n e i nthe Presence of Calcium................................. 88

17. Change in t h e UV S p e c t r u m o f 8 M U rea Used f o r Osmotic Shock as a F u n c t i o n o f Time o fIm m ers ion o f Root Samples ....................................................................... 99

18. Compari son o f OSM R e l e a s e d by Urea and M a n n i t o lT r e a t m e n t .............................................................................................................. 101

19. Comparison of the UV Absorption Spectra of BSA and OSM from Urea and Mannitol TreatmentSolutions .............................................................................................................. 103

20. S p e c t r a l Changes o f M a n n i t o l S o l u t i o n A f t e r T r e a t m e n tof Maize and Soybean Roots ..................................................................... 106

21. Elution Profile of Maize OSM from G-100 Sephadex ............... 108

22. Elution Profile of Dialyzed OSM Eluted from a G-50Sephadex Column ................................................................................................ 113

23. S t i m u l a t i o n o f PDE by P u r i f i e d OSM C a l m o d u l i n ...................... 125

24. Assay Procedure for Calmodulin .......................................... 137

25. S t a n d a r d Curve f o r C a l f B r a i n C a lm o d u l in .................................. 163

26. S t a n d a r d Curve f o r S p i n a c h L e a f C a lm o d u l in .............................. 165

27. Standard Curve for Maize Root Calmodulin .................................. 167

28. Com par i son o f S t i m u l a t i o n o f PDE by M aize , S p i n a c hand Bovine Calmodulin ........................................................................ 169

x

LIST OF PLATES

Plate

I . Growth Chamber and A p p a r a t u s f o r Measurement o f P l a s t i cand E l a s t i c Components o f Maize Root C e l lW all M a t e r i a l .................................................................................................... 21

I I . A t t a c h m e n t o f Maize Roo t t o T r a n s d u c e r A r m ........................... 24

I I I . A p p l i c a t i o n o f T e n s i o n t o Maize R oo t s ........................................... 26

IV. V i s u a l i z a t i o n o f P r o t e i n P r e s e n t i n OSM ...................................... 117

V. A ssa y f o r C a lm o d u l in C o n t e n t i n Maize OSM ................................. 120

VI. SDS-PAGE V i s u a l i z a t i o n o f P u r i f i e d OSM C a lm o d u l in .............. 123

V II . Comparison of Maize, Spinach and Bovine CalmodulinU s in g SDS-PAGE .................................................................................................. 146

V I I I . Maize C a lm o d u l in P u r i f i e d on a P h e n o t h i a z i n e - A f f i n i t yColumn Compared w i t h t h e Sample Loaded o n t o t h e Column ............................................................................................................ 149

IX. The Electrophoretic Mobility of Maize, Spinach, andBovine C a l m o d u l i n ........................................................................................... 154

X. P r o g r e s s i v e P u r i f i c a t i o n o f S p i n a c h L e a f C a lm o d u l in :S i n g l e S t a i n i n g ................................................................................................ 156

XI. P r o g r e s s i v e P u r i f i c a t i o n o f S p i n a c h L e a f C a lm o d u l in :Double S t a i n i n g ................................................................................................ 158

x i

LIST OF ABBREVIATIONS

MCE : f l - m e r c a p t o e t h a n o l ( a l s o l i s t e d as fl-MCE)

CaM : C a lm o d u l in

CPZ : C h l o r p r o m a z i n e

CWL : C e l l w a l l l o o s e n i n g

DMSO: D im e thy l s u l f o x i d e

EDTA: E t h y l e n e d i a m i n e t e t r a a c e t a t e , d i s o d i u m s a l t

EGTA: E t h y l e n e g l y c o l - b i s - ( - a m i n o e t h y l e t h e r ) -N , N , N ' , N ' - t e t r a c e t a t e d i s o d i u m s a l t

HEPES: N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c a c i d

MES : 2 - ( N - m o r p h o l i n o ) e t h a n e s u l f o n i c a c i d

MOPS: 3 - ( N - m o r p h o l i n o ) p r o p a n e s u l f o n i c a c i d

OSM : Osm oti c s h o c k m a t e r i a l

P-A : P h e n o t h i a z i n e a f f i g e l

PDE : P h o s p h o d i e s t e r a s e

PMSF: P h e n y l m e t h y l s u l f o n y l f l u o r i d e

TCA : T r i c h l o r o a c e t i c a c i d

TFP : T r i f l u o p e r a z i n e

T r i s : T r i s ( h y d r o x y r a e t h y l ) - a m i n o m e t h a n e

x i i

Introduction

P l a n t c e l l g ro w th i s most o f t e n d e s c r i b e d as an i r r e v e r s i b l e

i n c r e a s e i n c e l l volume ( C l e l a n d , 1 9 6 7 b ) . C e l l e n l a r g e m e n t r e q u i r e s

u p t a k e o f w a t e r and a d j u s t m e n t o f t h e i n t e r n a l s o l u t e c o n t e n t

( o s m o r e g u l a t i o n ) t o a c c o u n t f o r d i l u t i o n of i n t e r n a l s o l u t e s .

I r r e v e r s i b l e e n l a r g e m e n t f u r t h e r r e q u i r e s a l t e r a t i o n s o f t h e p l a n t

c e l l w a l l m a t r i x . Such a l t e r a t i o n i n v o l v e s b o t h t h e b r e a k i n g o f

e x i s t i n g bonds ( c e l l w a l l l o o s e n i n g ) and the a d d i t i o n o f new c e l l w a l l

m a t e r i a l . C e l l w a l l l o o s e n i n g r e s u l t s f rom c h a n g e s i n t h e b o n d in g

p a t t e r n s b e tw e e n t h e p e c t i c , h e m i c e l l u l o s i c , and c e l l u l o s i c component s

o f t h e c e l l w a l l . These c h a n g e s have b e e n examined on a macro s c a l e

by m e a s u r i n g t h e c h a n g e s i n t h e e l a s t i c i t y and p l a s t i c i t y o f p l a n t

t i s s u e (Tagawa and B onne r , 1957 ) , and t h e a l g a e N i t e l l a

( M e t r a u x e t a l . , 1980) and V a l o n i a ( T e p f e r and C l e l a n d , 1979 ) . The

t e rm s ' c e l l w a l l e x t e n s i b i l i t y ' and ' c e l l w a l l l o o s e n i n g ' a r e o f t e n

u s e d i n t e r c h a n g e a b l y and a r e e q u a t e d w i t h i n c r e a s e d c e l l w a l l

p l a s t i c i t y . Most m easu rem en ts of e l a s t i c i t y / p l a s t i c i t y have b e e n made

by a p p l i c a t i o n o f a u n i l a t e r a l t e n s i o n i n t h e l o n g t i t u d i n a l d i r e c t i o n

o f t h e t i s s u e (R a y le e t a l . , 1970; R a y le and C l e l a n d 1972a;

S t e u d l e e t a l . , 19 7 7 ) . E s t i m a t i o n o f c e l l w a l l e x t e n s i b i l i t y u s i n g

m u l t i a x i a l t e n s i o n h a s b e e n r e p o r t e d f o r the c e l l s o f V a l o n i a

( M e t r a u x e t a l . , 1980) and N i t e l l o p s i s ( S t e u d l e e t a l . , 1977) and th e

2

l a r g e e p i d e r m a l b l a d d e r c e l l s o f Mesembryantheman c r y s t a l l i n a m

(Zimmermann, 1978) .

P l a n t c e l l e n l a r g e m e n t was shown t o be s t i m u l a t e d by t h e p l a n t

hormone, i n d o l e - 3 - a c e t i c a c i d (IAA, a u x in ) by Went ( 1 9 3 4 ) , Thimann

(1936) and more r e c e n t l y i n v e s t i g a t e d by R a y le and C l e l a n d ( 1 9 7 0 ) .

The mechanism o f a u x i n - i n d u c e d g row th i s n o t known. The t h e o r y of

a u x in a c t i o n on g ro w th which h a s b e e n most t h o r o u g h l y i n v e s t i g a t e d i s

the a c i d g row th h y p o t h e s i s which was f o r m u l a t e d by R a y le and C l e l a n d

(1970) and Hage r e t a l . ( 1 9 7 1 ) . The g e n e r a l c o n c e p t p r o p o s e d by t h i s

h y p o t h e s i s i s t h a t t h e i n i t i a l a c t i o n o f a u x i n i s t o i n d u c e

a c i d i f i c a t i o n o f the c e l l w a l l by s t i m u l a t i n g H+ e f f l u x from th e

c y t o p l a s m i n t o t h e w a l l . These H+ a r e presumed t o f u n c t i o n as

s e c o n d a r y m e s s e n g e r s b y l o w e r i n g t h e c e l l w a l l pH and i n d u c i n g c e l l

w a l l l o o s e n i n g (CWL) t h r o u g h a c t i v a t i o n o f a CWL enzyme o r complex of

enzymes. A c i d i f i c a t i o n o f t h e w a l l h a s a l s o b e e n s u g g e s t e d t o induce

c e l l w a l l l o o s e n i n g by d i r e c t a c t i o n upon t h e components o f t h e c e l l

w a l l e i t h e r by h y d r o l y s i s o r c a t i o n d i s p l a c e m e n t . A l b e r s h e i m (1974)

and V a l e n t and A l b e r s h e i m (1974) have p r e s e n t e d e v i d e n c e c o n t r a d i c t o r y

t o t h e l a t t e r model . U s in g a model o l i g o s a c c h a r i d e s y s t e m , t h e y

showed t h a t d i r e c t h y d r o l y s i s o f c e l l w a l l component s a t t h e pH

e s t i m a t e d t o be e s t a b l i s h e d w i t h i n t h e w a l l ( c a . 5 . 0 ) i s p r o b a b l y

i n s u f f i c i e n t t o a c c o u n t f o r t h e o b s e r v e d change i n p l a s t i c i t y ( c e l l

w a l l l o o s e n i n g ) .

3

The a c i d g rowth h y p o t h e s i s p r e d i c t s t h a t exogenous a c i d i f i c a t i o n

o f t h e t i s s u e by a p p l i c a t i o n o f a c i d w i l l i n c r e a s e CWL and s t i m u l a t e

g r o w th . S t i m u l a t i o n o f g rowth by a p p l y i n g a c i d was d e s c r i b e d by

Bonner ( 1 9 3 4 ) . Avena c o l e o p t i l e t i s s u e segm en ts t r e a t e d w i t h pH 4 . 1

b u f f e r e x h i b i t e d a r a p i d enhancem en t (up t o 8 f o l d ) o f t h e i r

p r e - t r e a t m e n t g row th r a t e . The l a g t ime f o r a c i d s t i m u l a t e d g row th

( l e s s t h a n 1 min) i s c o n s i d e r a b l y s h o r t e r t h a n t h e t im e r e q u i r e d f o r

s t i m u l a t i o n o f g row th by a u x i n (7 - 15 m i n ) . R e - e x a m i n a t i o n o f a c i d

s t i m u l a t e d g rowth u s i n g s o l u t i o n s a c i d i f i e d by CC^ ( E va ns , 1967; R a y le

and C l e l a n d , 1972c) o r p h o s p h a t e b u f f e r s (R a y le and C l e l a n d , 1970)

s u g g e s t e d t h a t one f u n c t i o n o f a u x i n i s t o i n i t i a t e a c i d i f i c a t i o n o f

the c e l l w a l l .

The a c i d g rowth h y p o t h e s i s a l s o p r e d i c t s t h a t a u x i n - s t i m u l a t e d

g rowth w i l l be i n h i b i t e d by i n h i b i t i o n o f endogenous c e l l w a l l

a c i d i f i c a t i o n . Durand and R a y le (1973) and C l e l a n d and R a y le (1978)

d e m o n s t r a t e d such i n h i b i t i o n u s i n g p h o s p h a t e , c i t r a t e , and MES

b u f f e r s . T h a t exogenous a p p l i c a t i o n o f a u x i n i n d u c e s H+ s e c r e t i o n h a s

b e e n d e m o n s t r a t e d by C l e l a n d ( 1 9 7 3 ) . V espe r and Evans (1979) have

a l s o shown t h a t t i s s u e o f o a t and c o r n c o l e o p t i l e s w i l l lower t h e

e x t e r n a l pH o f t h e b a t h i n g medium (pH 5 . 2 - 5 . 5 ) d u r i n g g row th and t h a t

a u x i n - i n d u c e d H+ s e c r e t i o n can be n o n - h o r m o n a l l y s i m u l a t e d by

1 - n a p h t h y l a c e t a t e . V e s p e r and Evans p r o p o s e d t h a t n a p h t h y l a c e t a t e

i s r a p i d l y t a k e n up by c e l l s and h y d r o l y z e d by c y t o s o l i c e s t e r a s e s t o

r e l e a s e a c e t i c a c i d . The a c i d t h e n d i f f u s e s o u t o f t h e c e l l s i n t o t h e

c e l l w a l l , m im ick ing w a l l a c i d i f i c a t i o n i n d u c e d by a u x i n .

4

Auxin a p p l i e d t o g rowing t i s s u e s t i m u l a t e s b o t h a c i d i f i c a t i o n o f

t h e e x t e r n a l medium and g r o w th . A c i d i f i c a t i o n ( p re s u m a b l y by H+

s e c r e t i o n ) p r e c e d e s o b s e r v a b l e enhancem ent o f g ro w th , and by

c o m p a r i s o n , t h e d u r a t i o n o f a c i d s t i m u l a t e d g row th i s s h o r t e r t h a n

t h a t o f a u x i n - i n d u c e d g r o w th . T h i s s u g g e s t s t h a t a u x i n n o t o n l y

s t i m u l a t e s H+ s e c r e t i o n b u t a l s o i n i t i a t e s a d d i t i o n a l p r o c e s s e s

n e c e s s a r y f o r c o n t i n u e d g r o w th .

Auxin s t i m u l a t i o n o f H+s e c r e t i o n has b e e n p r o p o s e d t o be

r e g u l a t e d by a H+ ATPase pump l o c a l i z e d i n t h e p lasm a membrane

(Hage r e t a l . , 1 9 7 1 ) . A l th o u g h suc h an ATPase has n o t b e e n

i d e n t i f i e d , numerous p r o t e i n s a r e known t o be p r e s e n t w i t h i n t h e c e l l

w a l l and on t h e o u t e r s u r f a c e o f t h e p l a s m a membrane. One o f t h e

m a jo r p r o t e i n s found i n t h e c e l l w a l l o f d i c o t s i s a h y d r o x y p r o l i n e

r i c h p r o t e i n which has b e e n s u g g e s t e d t o f u n c t i o n as a r i g i d i f y i n g

component o f c e l l w a l l s (L a m por t , 1965; W i n t e r e t a l . , 1971;

C h r i s p e e l s , 1969, 1976; Cooper and V a r n e r , 1 9 8 1 ) . S e v e r a l c e l l w a l l

p r o t e i n s have b e e n shown t o p o s s e s s g l y c o l y t i c a c t i v i t y ( N e v in s ,

1 9 7 5 a ,b ) and may p l a y a r o l e i n t h e CWL p r o c e s s .

The extraction of cell wall proteins with minimal cytoplasmic

contamination has been a major difficulty. In bacterial systems,

osmotic shock has been used to release extra-cytoplasmic and plasma

membrane-associated proteins without disrupting the cells (Heppel,

1967; 1 9 6 9 ) . The proteins released include protein complexes which

traverse the membrane. Recently, Rubinstein (1977) reported that

osmotic shock of oat coleoptile tissue inhibits or abolishes both

a u x i n - i n d u c e d g rowth and H+ s e c r e t i o n . They r e p o r t e d t h a t o s m o t i c

shock , a l t h o u g h i n h i b i t i n g a u x i n s t i m u l a t e d a c i d i f i c a t i o n d i d n o t

i n h i b i t a u x i n - i n d u c e d c e l l w a l l l o o s e n i n g . Masuda e t a l . showed t h a t

o s m o t i c shoc k i n h i b i t i o n o f a u x i n - i n d u c e d a c i d i f i c a t i o n was r e v e r s i b l e

by 100 pM C a C ^ and c o n c l u d e d t h a t o s m o t i c shock does n o t a l t e r t h e

a u x i n - i n d u c e d g row th mechanism s i g n i f i c a n t l y . A d d i t i o n a l s t u d i e s by

R u b i n s t e i n (19 8 2 a ) c o n f i r m e d h i s o r i g i n a l f i n d i n g s t h a t o s m o t i c shoc k

r e l e a s e s numerous p r o t e i n s i n t o t h e medium b a t h i n g t h e t i s s u e . He

a l s o r e p o r t e d t h a t o s m o t i c shock i n h i b i t e d H+ s e c r e t i o n i n d u c e d by t h e

f u n g a l t o x i n f u s i c o c c i n ( R u b i n s t e i n , 198 2 b ) . T h i s t o x i n a p p e a r s t o

mimic t h e e f f e c t s o f a u x i n i n i t s a b i l i t y t o s t i m u l a t e b o t h H+

s e c r e t i o n and g r o w th . The a c t i o n o f t h e t o x i n d i f f e r s f rom t h a t o f

a u x in s i n c e s t i m u l a t i o n o f H+ s e c r e t i o n and g ro w th by f u s i c o c c i n i s

c o n s i d e r a b l y g r e a t e r t h a n t h a t in d u c e d by a u x i n (M a r rd , 1973; Yamagata

and Masuda, 1 9 7 5 ) . The a b i l i t y o f o s m o t i c s h o c k t o i n h i b i t

f u s i c o c c i n - i n d u c e d ( o r a u x i n - i n d u c e d ) H+ s e c r e t i o n s u g g e s t s t h a t an

e x t r a c e l l u l a r o r p lasm a m e m b r a n e - a s s o c i a t e d component n e c e s s a r y f o r

in d u ce d a c i d i f i c a t i o n i s r e l e a s e d by o s m o t i c sh o c k .

S e v e r a l mechanisms f o r low pH i n d u c t i o n o f c e l l w a l l l o o s e n i n g

have b e e n p r o p o s e d . They may be s e p a r a t e d i n t o t h r e e m a jo r

c a t e g o r i e s . One o f t h e e a r l i e s t p r o p o s a l s was t h a t d i s p l a c e m e n t o f

d i v a l e n t c a t i o n s from t h e c e l l w a l l m a t r i x ( u s u a l l y presumed t o be t h e

p e c t i c component ) by H+ c o u l d r e s u l t i n d e c r e a s e d v i s c o s i t y o f t h e

p e c t i c g e l s (Tagawa and B onne r , 1957 ) . T h i s model h a s b e e n d e s c r i b e d

as t h e c a l c i u m b r i d g e c r o s s - l i n k i n g c o n c e p t . R e c e n t l y , Kauss and

G l a z e r (1977) s u g g e s t e d a m o d i f i c a t i o n o f t h i s model i n which s p e c i f i c

d i s p l a c e m e n t o f c a l c i u m from a w a l l bound l e c t i n may d e c r e a s e or

i n a c t i v a t e t h e b i n d i n g o f t h e l e c t i n w i t h i n t h e c e l l w a l l . The

d e c r e a s e d b i n d i n g o f t h e l e c t i n , i n s t e a d o f a r e d u c t i o n i n t h e number

o f c a l c i u m b r i d g e s be tw e en t h e c e l l w a l l p o l y s a c c h a r i d e s , would r e s u l t

i n c e l l w a l l l o o s e n i n g . A s e c o n d mechanism f o r a c i d a c t i o n on t h e

c e l l w a l l m igh t be d i r e c t c e l l w a l l l o o s e n i n g by d i s r u p t i o n o f

e x i s t i n g h y d r o g e n bonds ( K e e g s t r a e t a l . , 1 9 7 3 ) . I o n i c b o nds ,

e s p e c i a l l y h y d r o g e n b o n d i n g , b e tw e en t h e c e l l u l o s e and h e m i c e l l u l o s e

component s o f t h e c e l l w a l l would be weakened and d i s r u p t e d a t low pH.

A t h i r d p r o p o s a l f o r t h e n a t u r e o f a c i d - i n d u c e d w a l l l o o s e n i n g i s t h a t

c e l l w a l l l o o s e n i n g i s e n z y m a t i c and t h a t a d e c r e a s e d c e l l w a l l pH

s t i m u l a t e s o r t r i g g e r s a c e l l w a l l l o o s e n i n g enzyme o r complex

(Hage r e t a l . , 1971 ) .

T e p f e r and C l e l a n d (1979) c o n c l u d e d t h a t t h e d i f f e r e n c e s be tw e en

V a l o n i a and o a t c o l e o p t i l e c e l l w a l l l o o s e n i n g s u g g e s t t h a t o a t

c o l e o p t i l e s may r e q u i r e e n z y m a t i c l o o s e n i n g w h i l e V a l o n i a e x h i b i t s a

more d i r e c t , p h y s i c a l a c t i o n o f low pH on t h e c e l l w a l l .

The p r e d o m i n a n t d i v a l e n t c a t i o n p r e s e n t i n p l a n t c e l l w a l l s i s

c a l c i u m . S e n t e n a c and G r ig n o n (1981 ) m ea s u red c e l l w a l l c a l c i u m by

e q u i l i b r i u m d i a l y s i s and m o n o v a le n t c a t i o n c o m p e t i t i o n . They r e p o r t e d

c a l c i u m ' c o n c e n t r a t i o n s as l a r g e as 0 . 6 2 5 m m o l . / g - d r y w e i g h t i n b r o a d

b ean ( V i c i a f a b a L . , v a r . M in o r ) and Y e l lo w L u p in e (L u p in u s

l u t e u s L . ) . The p r e v a l e n c e o f c a l c i u m i n t h e c e l l w a l l would s u g g e s t

t h a t some form o f c r o s s - l i n k i n g b e tw e en t h e c a t i o n and t h e c a r b o x y l

7

g ro u p s o f t h e u r o n i c a c i d r e s i d u e s i n t h e c e l l w a l l m ig h t c o n t r i b u t e

t o m a i n t e n a n c e o f c e l l w a l l r i g i d i t y . R e l e a s e o f t h e c a l c i u m c o u l d

t h e n a c c o u n t f o r i n c r e a s e d c e l l w a l l e x t e n s i b i l i t y . Fo r o a t

c o l e o p t i l e s , u n l e s s a c i d - i n d u c e d c a l c i u m r e l e a s e i s enzyme m e d i a t e d , a

w a l l l o o s e n i n g model b a s e d on a c i d - i n d u c e d c a l c i u m d i s p l a c e m e n t i s n o t

c o m p a t i b l e w i t h t h e r e s u l t s o f T e p f e r and C l e l a n d ( 1 9 7 9 ) . They have

r e p o r t e d t h a t EDTA can n o t s u b s t i t u t e f o r a c i d i n w a l l l o o s e n i n g and

t h a t w a l l l o o s e n i n g i s d e p e n d e n t upon w a l l p r o t e i n . I n t h e c a s e o f

a u x i n - i n d u c e d w a l l l o o s e n i n g , which may be a c i d m e d i a t e d , o n l y a s m a l l

f r a c t i o n o f t h e t o t a l c e l l w a l l c a l c i u m i s r e l e a s e d i n r e s p o n s e t o

a u x i n and t h e amount r e l e a s e d i s i n s u f f i c i e n t t o a c c o u n t f o r t h e

o b s e r v e d c e l l w a l l l o o s e n i n g ( C l e l a n d , 1 9 6 0 ) . I f t h e m a jo r r o l e o f

c e l l w a l l c a l c i u m i s t o m a i n t a i n t h e r i g i d i t y o f t h e w a l l , t h e n t h e

s m a l l f r a c t i o n o f c a l c i u m r e l e a s e d by a u x i n - t r e a t e d t i s s u e s compared

w i t h t h e a p p a r e n t l y h i g h c o n c e n t r a t i o n o f c a l c i u m i n t h e c e l l w a l l , as

m easu red by S e n t e n a c and G r ig n o n ( 1 9 8 1 ) , a p p e a r s i n c o n g r u o u s w i t h a

model f o r a u x i n a c t i o n b a s e d on m a s s i v e c a l c i u m d i s p l a c e m e n t f rom t h e

w a l l . T h i s d i s c r e p a n c y o f t o t a l c e l l w a l l c a l c i u m compared t o t h e

amount r e l e a s e d i n r e s p o n s e t o a u x i n s u g g e s t s t h a t w a l l c a l c i u m may

s e r v e some f u n c t i o n o t h e r t h a n a s t i f f e n i n g component o f t h e w a l l .

T e p f e r and T a y l o r (1981) examined t h e e f f e c t o f c a l c i u m upon p e c t i n

g e l l a t i o n and found t h a t t h e i n c r e a s e i n p e c t i n g e l r i g i d i t y in d u c e d

by c a l c i u m i s i n s u f f i c i e n t t o a c c o u n t f o r e i t h e r no rm a l o r

c a l c i u m - i n d u c e d c e l l w a l l s t i f f e n i n g . F u r t h e r m o r e , t h e

p l a s t i c / e l a s t i c c o m p l i a n c e o f c o l e o p t i l e ( C l e l a n d and R a y l e , 1977) o r

pea s t e m ( C o a r t n e y and M o rre , 1980) w a l l p r e p a r a t i o n s i s n o t a l t e r e d

by t h e a d d i t i o n o f c a l c i u m . However , c e l l w a l l p l a s t i c i t y can be

d e c r e a s e d by ^ n v i t r o t r e a t m e n t w i t h c a l c i u m i n t h e p r e s e n c e o f a c i d .

T h i s s u g g e s t s t h a t c a l c i u m may r e g u l a t e an a u x i n - d e p e n d e n t c e l l w a l l

s t i f f e n i n g p r o c e s s and does n o t a c t d i r e c t l y upon t h e c e l l w a l l .

C a lc iu m i n h i b i t i o n o f g rowth does n o t r e s u l t f rom a s i m p l e

r e d u c t i o n i n t h e t u r g o r o f t h e t i s s u e c a u se d by t h e o s m o t i c e f f e c t s o f

t h e a p p l i e d s o l u t i o n o f C a C ^ * C a lc iu m ( a s C a C ^ ) a t a c o n c e n t r a t i o n

o f 20 mM r a p i d l y i n h i b i t s o a t c o l e o p t i l e g r o w t h . M a n n i t o l , a t

e q u i v a l e n t o s m o t i c c o n c e n t r a t i o n s ( a b o u t 60 mM), has v i r t u a l l y no

e f f e c t . The abundance o f c a l c i u m i n t h e c e l l w a l l , t h e r e l a t i v e l y low

c o n c e n t r a t i o n o f c a l c i u m i n t h e c y t o p l a s m and t h e numerous p r o c e s s e s

known t o r e q u i r e , o r be m o d i f i e d by c a l c i u m s u g g e s t s t h a t t h e c a l c i u m

p r e s e n t i n t h e c e l l w a l l may be i n v o l v e d i n c e l l g ro w th and

d e v e lo p m en t i n a c a p a c i t y e x t e n d i n g beyond t h a t o f a c e l l w a l l

s t r u c t u r a l component . The o b s e r v a t i o n s o f C l e l a n d and R a y l e (1977)

and C o a r t n e y and Morre (1980) t h a t c a l c i u m does n o t d i r e c t l y a l t e r t h e

m e c h a n i c a l p r o p e r t i e s o f t h e c e l l w a l l , t h a t g row th i n h i b i t i o n by

c a l c i u m i s n o t t h e r e s u l t o f a s i m p l e t u r g o r r e d u c t i o n and t h a t

c a l c i u m t r e a t m e n t o f n o n - d e n a t u r e d f r o z e n / t h a w e d c e l l m a t e r i a l

d r a m a t i c a l l y i n h i b i t s a c i d - i n d u c e d CWL f u r t h e r s u g g e s t t h a t c a l c i u m

f u n c t i o n s as a r e g u l a t o r o f a u x i n - i n d u c e d g r o w th .

9

Numerous m e t a b o l i c and d e v e l o p m e n t a l p r o c e s s e s have b e e n r e p o r t e d

t o r e q u i r e c a l c i u m i n b o t h a n im a l and p l a n t s y s te m s ( C u t h b e r t , 1970;

S c a r p a and C a r a f o l i , 1978; Cheung, 1 9 8 0 ) . I n p l a n t s , t h e t r a n s p o r t

sy s te m f o r s e r i n e i s d e p e n d e n t upon and s t i m u l a t e d by c a l c i u m

(S m i th , 1 9 7 8 a , b ) . C a l c iu m and IAA i n d u c e o p p o s i t e ch a n g es i n t h e

t h i c k n e s s o f t h e p lasm a membrane (IAA d e c r e a s e s , c a l c i u m i n c r e a s e s

membrane t h i c k n e s s ) and a l t e r i t s m i c r o v i s c o s i t y (Morre and B r a c k e r ,

1976; H e l g e r s o n e t a l . , 1976 ) . D e la F u e n t e and L e o p o ld (1973)

d e m o n s t r a t e d a s p e c i f i c r e q u i r e m e n t f o r c a l c i u m i n t h e p o l a r t r a n s p o r t

o f a u x i n and s u g g e s t e d t h a t c a l c i u m e i t h e r b i n d s t o t h e a u x i n

t r a n s p o r t r e c e p t o r o r i s c o - t r a n s p o r t e d w i t h a u x i n . Two s p e c i f i c

s t u d i e s have d e m o n s t r a t e d t h e i n v o l v e m e n t o f c a l c i u m i n p r o t e i n

b i n d i n g o r a c t i v a t i o n . LeJohn e t a l . (1974) d e m o n s t r a t e d t h a t

c y t o k i n i n s t i m u l a t e s t h e r e l e a s e o f c a l c i u m f rom a c e l l w a l l l o c a l i z e d

g l y c o p r o t e i n i n t h e oomycete A c h l y a . C y t o k i n i n f u r t h e r s t i m u l a t e s t h e

u p t a k e o f c a l c i u m from t h e medium i n t h i s o r g a n i s m . Kauss (1981) has

r e p o r t e d t h a t o s m o r e g u l a t i o n i n P o t e r i o o c h r o m o n a s m a lh a m e n s i s i s

d e p e n d e n t upon ch a n g es i n t h e c o n c e n t r a t i o n o f t h e f l o r i d i a n s t a r c h

a n a l o g u e , i s o f l o r i s i d e . The enzyme i s o f l o r i s i d e p h o s p h a t e s y n t h a s e ,

which r e g u l a t e s t h e c o n c e n t r a t i o n o f t h i s p o l y s a c c h a r i d e , i s a c t i v a t e d

s p e c i f i c a l l y by c a l c i u m .

C a lc iu m has b e e n r e p o r t e d t o i n h i b i t t h e g row th o f p e a s tem

se gm e n ts ( B u r s t r o m , 1964; Ray, 1 9 6 2 ) , o a t c o l e o p t i l e s (Tagawa and

B onne r , 1 9 5 7 ) , and bean s t e m segm en ts ( T e p f e r and T a y l o r , 1981) a t

c o n c e n t r a t i o n s be tw e en 5 and 20 mM. P o o v i a h and L eopo ld (1976)

r e p o r t e d t h a t c a l c i u m (1 mM C a C ^ ) d e c r e a s e s a u x i n - i n d u c e d g r o w th o f

s oybe an h y p o c o t y l s by 50%. I n t h e same s t u d y , 1 mM c a l c i u m was shown

t o i n c r e a s e a u x i n b i n d i n g t o p e l l e t a b l e membrane f r a c t i o n s .

A u x i n - in d u c e d a c i d i f i c a t i o n o f t h e e x t e r n a l medium was r e p o r t e d

by Cohen and N a d l e r (1976 ) t o be s t i m u l a t e d by 0 . 3 - 1 mM c a l c i u m .

They s u g g e s t e d t h a t c a l c i u m s p e c i f i c s t i m u l a t i o n o f a u x i n m e d i a t e d

• • +2 + a c i d i f i c a t i o n i n d i c a t e s t h a t a Ca /H -ATPase t r a n s p o r t s y s t e m may be

i n v o l v e d in a u x i n - i n d u c e d a c i d i f i c a t i o n . One may n o t e t h a t t h e

c o n c e n t r a t i o n s o f c a l c i u m u s e d i n a l l o f t h e above s t u d i e s i s

c o n s i d e r a b l y g r e a t e r t h a n t h e presumed c y t o s o l i c c a l c i u m

c o n c e n t r a t i o n . E x t r a p o l a t i n g f rom m easu rem en ts made f o r a n im a l

sy s te m s (C obbo ld , 1979) , t h e a n t i c i p a t e d f r e e c a l c i u m c o n t e n t o f t h e

c y t o p l a s m would be i n t h e n a n o - o r m ic r o m o l a r r a n g e . T h i s i n d i c a t e s

t h a t a t l e a s t one m a jo r e f f e c t o f exogenous c a l c i u m would be t o

p e r t u r b t h e c a r e f u l l y r e g u l a t e d p h y s i o l o g i c a l e q u i l i b r i u m b e tw e en f r e e

c y t o p l a s m i c and bound ( o r s e q u e s t e r e d ) c a l c i u m .

The d i v e r s e r o l e s o f c a l c i u m s u g g e s t s t h a t , as i n a n im a l s y s t e m s ,

c a l c i u m i n p l a n t s may f u n c t i o n as a s e c o n d a r y m e s s e n g e r . The

a l t e r a t i o n o f a u x i n - i n d u c e d g r o w t h by c a l c i u m f u r t h e r i m p l i c a t e s t h i s

c a t i o n a s a m e s s e n g e r o r r e g u l a t o r y component o f t h e mechanism o f

a u x i n a c t i o n in p l a n t g r o w t h . D u r in g t h e p a s t d e c a d e , t h e

11

r o l e o f c a l c i u m as a m e s s e n g e r ha s been p a r t i a l l y c l a r i f i e d by t h e

i d e n t i f i c a t i o n o f s e v e r a l p r o t e i n s which b i n d and a r e c o n t r o l l e d by

c a l c i u m . The r e g u l a t i o n by c a l c i u m o f b o t h m u s c u l a r c o n t r a c t i o n and

t h e e p i n e p h r i n e - s t i m u l a t e d a m p l i f i c a t i o n c a s c a d e have b e e n i n t e n s e l y

s t u d i e d . R e g u l a t i o n o f b o t h s y s te m s i n v o l v e s a m o d u l a t o r p r o t e i n

which f u n c t i o n s as t h e r e c e p t o r o f t h e s e c o n d a r y m e s s e n g e r c a l c i u m .

Two g r o u p s (Cheung, 1967; Cheung, 1971; K a k i u c h i e t a l . , 1973)

r e p o r t e d t h e i s o l a t i o n , f rom r a t b r a i n , o f a s m a l l p r o t e i n p o s s e s s i n g

no a p p a r e n t e n z y m a t i c a c t i v i t y o r s t r u c t u r a l r o l e . T h i s p r o t e i n was

o b s e r v e d t o s t i m u l a t e t h e d e g r a d a t i o n o f 3 * : 5 1 cAMP by t h e enzyme

p h o s p h o d i e s t e r a s e (PDE). T h i s a c t i v a t o r has s i n c e b e e n named

c a l m o d u l i n (CaM) and has b e e n found t o be p r e s e n t i n e v e r y e u k a r y o t e

exam ined . C a l m o d u l in , i n a n im a l s y s t e m s , a p p e a r s t o be i n v o l v e d i n

v i r t u a l l y e v e r y p r o c e s s known t o r e q u i r e c a l c i u m . Those i n s t a n c e s f o r

which no r o l e o f c a l m o d u l i n h a s b e e n d e m o n s t r a t e d have b e e n shown t o

i n v o l v e one o f t h e l a r g e r g ro u p o f c a l c i u m m o d u l a t o r p r o t e i n s ( S -1 0 0 ,

p a r v a l b u m i n , t r o p o n i n - l ) .

The u b i q u i t y o f CaM i n a n im a l s y s t e m s would c e r t a i n l y s u g g e s t

t h a t CaM m igh t f u n c t i o n as a m o d u l a t o r o f c a l c i u m d e p e n d e n t mechanisms

i n p l a n t s . C a lm o d u l in has b e e n i s o l a t e d from s p i n a c h l e a v e s

( W a t t e r s o n e t a l . , 1980) and shown t o d i f f e r s l i g h t l y f rom a n im a l

c a l m o d u l i n s . The i n v o lv e m e n t o f c a l m o d u l i n i n t h e r e g u l a t i o n o f

g rowth and g r a v i t r o p i s m has b e e n s u g g e s t e d (Roux and S locum, 19 8 3 ) .

R e c e n t l y , E l l i o t (1980) r e p o r t e d t h a t c y t o k i n i n s t i m u l a t i o n o f

b e t a c y a n i n s y n t h e s i s i s i n h i b i t e d by t h e c a l m o d u l i n i n h i b i t o r

12

t r i f l u o p e r a z i n e (TFP) . D i e t e r and Marme (1980) found t h a t u p t a k e o f

c a l c i u m by a m ic ro so m a l f r a c t i o n i s s t i m u l a t e d i n v i t r o by CaM, w h i l e

Slocum and Roux (1983) have r e p o r t e d t h e p o s s i b l e i n v o lv e m e n t o f CaM

i n t h e r e g u l a t i o n o f g r a v i t r o p i c r e s p o n s e s . The o b s e r v a t i o n s o f

Lee e t a l . (1983) t h a t g r a v i t r o p i s m i s s t r o n g l y d e p e n d e n t upon t h e

+2d e v e lopm en t o f a t r a n s v e r s e Ca g r a d i e n t i n r o o t t i p s o f m a ize

s u p p o r t t h e i n t r i g u i n g p o s s i b i l i t y o f CaM m o d u l a t i o n o f g r a v i t r o p i c

r e s p o n s e s . A nderson and C orm ie r (1978) d e m o n s t r a t e d t h a t NAD k i n a s e

i s c a l c i u m d e p e n d e n t and r e g u l a t e d by c a l m o d u l i n . C a lm o d u l in r e c e n t l y

has b e e n r e p o r t e d t o be p r e s e n t w i t h i n t h e c h l o r o p l a s t

( J a r r e t t e t a l . , 1 9 8 2 ) . The p r e s e n c e o f CaM w i t h i n t h e c h l o r o p l a s t

poses t h e i n t r i g u i n g q u e s t i o n o f w h e t h e r i t i s s y n t h e s i z e d by o r

t r a n s p o r t e d i n t o t h e c h l o r o p l a s t . A l th o u g h CaM i s known t o b i n d t o

t h e o u t e r membrane o f m i t o c h o n d r i a , i t has n o t b e e n i d e n t i f i e d w i t h i n

t h i s o r g a n e l l e .

The e v i d e n c e t h a t c a l m o d u l i n i s p r e s e n t i n p l a n t s and may be

i n v o l v e d i n t h e mechan isms o f g rowth and g r a v i t r o p i s m s u g g e s t s t h a t

a u x i n - i n d u c e d g row th may i n v o l v e t h e i n t e r a c t i o n o f a u x i n , H+ ,

c a l c i u m , and c a l m o d u l i n . The n e c e s s i t y o f c a l c i u m f o r r o o t g row th was

d e m o n s t r a t e d by B u r s t r o m ( 1 9 6 4 ) , a l t h o u g h t h e r e q u i r e m e n t o f c a l c i u m as

a p l a n t n u t r i e n t was known p r e v i o u s l y . B u r s t r o m d e m o n s t r a t e d n o t o n l y

t h a t c a l c i u m d e f i c i e n c y i n h i b i t e d r o o t g r o w th b u t a l s o t h a t r o o t

g rowth was s t i m u l a t e d by low (1 mM) and i n h i b i t e d by h i g h (20 mM)

c o n c e n t r a t i o n s o f c a l c i u m .

13

The e l o n g a t i o n zone o f r o o t s i s h i g h l y s e n s i t i v e t o IAA. Thimann

(1936) r e p o r t e d t h a t s t i m u l a t i o n o f r o o t g row th by a u x i n e x h i b i t e d a

d osage r e s p o n s e c u r v e s i m i l a r t o t h a t o f s tem and c o l e o p t i l e s e c t i o n s

e x c e p t t h a t t h e e n t i r e c u r v e f o r r o o t s was s h i f t e d t o a lower

c o n c e n t r a t i o n r a n g e . Both c o l e o p t i l e and s tem t i s s u e e x h i b i t maximal

s t i m u l a t i o n by a u x i n a t c o n c e n t r a t i o n s o f 1 - 10 pM. By c o n t r a s t ,

o p t i m a l s t i m u l a t i o n o f r o o t g row th was r e p o r t e d t o o c c u r a t

c o n c e n t r a t i o n s o f 0 . 1 nM, w i t h s e v e r e i n h i b i t i o n a t 1 uM (Thimann,

1936 ) . S t i m u l a t i o n o f r o o t g row th by a u x i n has been c o n t r o v e r s i a l

p r i m a r i l y b e c a u s e d e m o n s t r a t i o n o f s u c h s t i m u l a t i o n i s n o t e n t i r e l y

r e p e a t a b l e . R e c e n t l y , Mulkey e t a l . ( 1982a ) r e p o r t e d t h a t s t r o n g

s t i m u l a t i o n (up t o 400 %) o f i n t a c t m a ize r o o t g rowth by 0 . 1 - 1 . 0 nM

a u x i n c a n be r e p r o d u c i b l y o b t a i n e d p r o v i d e d t h e r o o t s a r e p r e t r e a t e d

w i t h t h e e t h y l e n e i n h i b i t o r s c o b a l t and AVG ( a m i n o e t h o x y v i n y l g l y c i n e ) .

T h e i r f i n d i n g s f u r t h e r s u g g e s t t h a t t h e p ro b le m o f poor

r e p r o d u c i b i l i t y o f r o o t g row th s t i m u l a t i o n by a u x i n r e s u l t s f rom

a u x i n - i n d u c e d e t h y l e n e p r o d u c t i o n w i t h i n t h e r o o t a t low

c o n c e n t r a t i o n s o f a u x i n .

The a c i d g rowth h y p o t h e s i s a p p e a r s a p p l i c a b l e t o t h e g row th o f

r o o t s . Edwards and S c o t t (1974) and McBride and Evans (1977) have

d e m o n s t r a t e d t h a t m a ize and l e n t i l r o o t g rowth i s s t i m u l a t e d by a c i d

(pH 4 . 0 ) . McBride and Evans (1977) and Mulkey e t a l . (1982b) have

a l s o r e p o r t e d t h a t b o t h l e n t i l and m a i z e r o o t s a c i d i f y t h e b a t h i n g

medium d u r i n g normal g r o w th , w h i l e Evans e t a l . ( 1980) have

d e m o n s t r a t e d t h a t m a ize r o o t s r a i s e t h e pH o f t h e b a t h i n g medium i n

14

t h e p r e s e n c e o f g r o w t h - i n h i b i t i n g c o n c e n t r a t i o n s o f a u x i n . Mulkey and

Evans (1981 ) have p r o v i d e d d i r e c t v i s u a l e v i d e n c e o f H+ s e c r e t i o n by

i n t a c t m a iz e r o o t s u s i n g t h e a g a r dye method d e s c r i b e d by

W e i s e n s e e l e t a l . ( 1979) and i t has been shown t h a t c o n c e n t r a t i o n s o f

a u x in which s t i m u l a t e r o o t g row th a l s o s t i m u l a t e H+ e f f l u x from r o o t s

(Mulkey e t a l . , 1982b) when e t h y l e n e s y n t h e s i s i s i n h i b i t e d .

I n s p i t e o f t h e s u b s t a n t i a l c i r c u m s t a n t i a l e v i d e n c e i n d i c a t i n g

i n t e r a c t i v e r o l e s f o r a u x i n , c a l c i u m , and H+ i n t h e r e g u l a t i o n o f r o o t

e l o n g a t i o n , ou r u n d e r s t a n d i n g o f t h e n a t u r e o f t h e g row th p r o c e s s i n

r o o t s i s i n c o m p l e t e . I t i s c l e a r t h a t e a r l i e r models s u g g e s t i n g a u x i n

c o n t r o l o f g row th by m o d i f i c a t i o n o f w a l l p l a s t i c i t y t h r o u g h

d i s p l a c e m e n t o f c a l c i u m o r m o d i f i c a t i o n o f t h e d r i v i n g f o r c e b e h i n d

g rowth by o s m o t i c a d j u s t m e n t a r e i n s u f f i c i e n t t o e x p l a i n t h e mechanism

o f a c t i o n o f a u x i n . With t h e i n c r e a s i n g e v i d e n c e f o r c r i t i c a l r o l e s

o f c a l c i u m and low pH i n t h e w a l l l o o s e n i n g p r o c e s s , a number o f

i m p o r t a n t q u e s t i o n s a r i s e r e g a r d i n g t h e r o l e s t h a t t h e s e r e g u l a t o r s

p l a y i n t h e a c t i o n o f a u x i n a t t h e m o l e c u l a r l e v e l . The work

p r e s e n t e d i n t h i s d i s s e r t a t i o n i s f o c u s e d on t h e f o l l o w i n g q u e s t i o n s ;

(1 ) What a r e t h e p h y s i c a l c h a r a c t e r i s t i c s o f m a ize r o o t c e l l w a l l s and

i n what r e s p e c t does t h i s t i s s u e d i f f e r f rom c e l l w a l l m a t e r i a l o f o a t

c o l e o p t i l e s and t h e c e l l s o f V a l o n i a and N i t e l l a ? ( 2 ) What i s t h e

s h o r t t e r m e f f e c t o f i n c r e a s e d c a l c i u m c o n c e n t r a t i o n upon r o o t g row th?

( 3 ) To wha t e x t e n t i s a c i d - i n d u c e d g ro w th d e p e n d e n t upon components

r e l e a s e d f rom r o o t t i s s u e e x p o s e d t o o s m o t i c shock? ( 4 ) I s t h e r e a

c a l c i u m i n t e r a c t i v e component o f t h e o s m o t i c shoc k r e l e a s e m a t e r i a l ?

(5 ) I s c a l m o d u l i n p r e s e n t i n m a iz e r o o t s ? and (6 ) I f c a l m o d u l i n

p r e s e n t e i t h e r i n t h e c y t o p l a s m o r t h e o s m o t i c shock m a t e r i a l ,

p r o t e i n ( s ) may i n t e r a c t w i t h CaM?

C h a p t e r I . M easurement o f t h e P l a s t i c and E l a s t i c C om pl iance o f Maize

Root T i s s u e and t h e I n d u c t i o n o f C e l l Wall L o o s e n in g by Ac id and EDTA.

I n t r o d u c t i o n .

The p l a n t c e l l w a l l i s a dynamic s t r u c t u r e . D u r in g g ro w th , t h e

w a l l must u n d e rg o i r r e v e r s i b l e ( n o n - e l a s t i c ) e x p a n s i o n and a d d i t i o n a l

c e l l w a l l m a t e r i a l must be a dded . The p l a n t w a l l i s g e n e r a l l y

c o n s i d e r e d t o c o n t a i n t h r e e m a jo r c a t e g o r i e s o f p o l y s a c c h a r i d e

components and s e v e r a l p r o t e i n s . The p o l y s a c c h a r i d e components a r e

c a t e g o r i z e d as c e l l u l o s e , h e m i c e l l u l o s e , and p e c t i n and a r e commonly

d e s c r i b e d as t h e cement ( h e m i c e l l u l o s e and p e c t i n ) and s t r u c t u r a l

f ramework ( c e l l u l o s e m i c r o f i b r i l s ) o f t h e w a l l . I r r e v e r s i b l e

e x p a n s i o n o f t h e c e l l w a l l a p p e a r s t o r e q u i r e b r e a k a g e and r e f o r m i n g

o f bonds i n t h e p r e s e n c e o f t e n s i o n . I n t h e l i v i n g s y s t e m , t h e

t e n s i o n i s m u l t i a x i a l and p r o v i d e d by t u r g o r d e v e lo p e d w i t h i n t h e

c e l l . C l e l a n d (1958) has r e p o r t e d t h a t c e l l w a l l s o f f r o z e n / t h a w e d

( F /T ) o a t c o l e o p t i l e s e x h i b i t i n c r e a s e d e x t e n s i o n , u n d e r t e n s i o n , i f

p r e - t r e a t e d w i t h a u x i n p r i o r t o k i l l i n g t h e t i s s u e . C l e l a n d ( 1 9 6 7 a )

and C l e l a n d and R a y l e (1977) have r e p o r t e d t h a t a c i d t r e a t m e n t o f F/T

Avena c o l e o p t i l e t i s s u e i n c r e a s e s t h e c e l l w a l l e x t e n s i b i l i t y .

C l e l a n d and R a y le have a l s o shown t h a t a c i d - i n d u c e d e x t e n s i o n o f F /T

t i s s u e i s r e v e r s i b l y i n h i b i t e d by c a l c i u m . These f i n d i n g s s u g g e s t

16

t h a t t h e mechan ism o f c o l e o p t i l e c e l l w a l l e x p a n s i o n r e q u i r e s H+ f o r

c e l l w a l l l o o s e n i n g (CWL), and t h a t a u x i n - i n d u c e d CWL i s m e t a b o l i c a l l y

d e p e n d e n t . The a b i l i t y o f c a l c i u m t o i n h i b i t a c i d - i n d u c e d e x t e n s i o n

o f F /T t i s s u e s u g g e s t s t h a t c a l c i u m m e d i a t i o n o f CWL may be i n v o l v e d .

T e p f e r and C l e l a n d (1979) compared t h e c h a r a c t e r i s t i c s o f c e l l w a l l

e x t e n s i o n o f o a t c o l e o p t i l e s and V a l o n i a . The t i s s u e was k i l l e d by

f r e e z e / t h a w c y c l e s which d i s r u p t e d t h e p r o t o p l a s t . T i s s u e so t r e a t e d

was c o n s i d e r e d " c e l l w a l l m a t e r i a l " , s i n c e t h e membrane components o f

t h e c e l l s were no l o n g e r f u n c t i o n a l . C e l l w a l l m a t e r i a l o f V a l o n i a

and Avena were m e a s u r e d f o r a c i d - i n d u c i b l e e x t e n s i o n and were found t o

r e s p o n d s i m i l a r l y t o a c i d t r e a t m e n t when m a i n t a i n e d u n d e r a p p l i e d

t e n s i o n . However , V a l o n i a e x h i b i t e d a l a r g e a c i d - i n d u c i b l e e x t e n s i o n

i n t h e a b s e n c e o f t e n s i o n , which was n o t o b s e r v e d f o r o a t c o l e o p t i l e s .

The c e l l w a l l o f V a l o n i a a l s o e x h i b i t e d a l a r g e i n c r e a s e i n

e x t e n s i b i l i t y when t r e a t e d w i t h e i t h e r EDTA o r EGTA. A l th o u g h o a t

c o l e o p t i l e c e l l w a l l e x t e n s i b i l i t y was a l s o i n c r e a s e d by t h e s e

d i v a l e n t c a t i o n c h e l a t o r s , t h e m a g n i tu d e o f t h e e x t e n s i o n was o n l y

10 - 20 % as l a r g e as t h a t i n d u c e d by a c i d t r e a t m e n t and was much

s m a l l e r t h a n t h e EDTA e f f e c t upon V a l o n i a c e l l w a l l s . A c i d - i n d u c e d

e x t e n s i o n i n b o t h V a l o n i a and Avena was i n h i b i t e d when c a l c i u m was

i n c l u d e d i n t h e a c i d i c b u f f e r . Removal o f t h e c a l c i u m r e s u l t e d i n

no rm a l a c i d - i n d u c e d e x t e n s i o n a f t e r a s h o r t l a g p e r i o d o f a few

m i n u t e s .

18

P r o t e i n d e n a t u r i n g t r e a t m e n t s a b o l i s h e d a c i d - i n d u c e d e x t e n s i o n i n

o a t c o l e o p t i l e s b u t n o t i n V a l o n i a . T he se f i n d i n g s s u g g e s t m a jo r

d i f f e r e n c e s be tw e en o a t c o l e o p t i l e and V a l o n i a c e l l w a l l e x t e n s i o n

mechanisms and i n d i c a t e t h a t V a l o n i a c e l l w a l l l o o s e n i n g i s p r i m a r i l y

d e p e n d e n t upon t h e d i s p l a c e m e n t o f d i v a l e n t c a t i o n s , mos t p r o b a b l y

c a l c i u m , from t h e c e l l w a l l . I n c o n t r a s t , t h e i n a b i l i t y o f EDTA t o

mimic a c i d - i n d u c e d e x t e n s i o n i n Avena c o l e o p t i l e s and t h e l o s s o f

a c i d - i n d u c i b l e c e l l w a l l l o o s e n i n g a f t e r p r o t e i n d e n a t u r a t i o n s u g g e s t s

t h a t o a t c o l e o p t i l e w a l l l o o s e n i n g i s e n z y m a t i c . F u r t h e r , t h e

i n h i b i t i o n by c a l c i u m o f a c i d - i n d u c e d e x t e n s i o n s u g g e s t s t h a t

e n z y m a t i c c e l l w a l l l o o s e n i n g i n Avena may be r e g u l a t e d by o r a t l e a s t

r e q u i r e c a l c i u m .

McBride and Evans (1977) d e s c r i b e d t h e r a p i d s t i m u l a t i o n o f i n t a c t

l e n t i l r o o t s by a c i d . Edwards and S c o t t ( 1974) r e p o r t e d a r a p i d

s t i m u l a t i o n o f e l o n g a t i o n o f m a ize r o o t segm en ts by low pH. The

r e s p o n s e o f r o o t s d i f f e r s f rom t h a t o f c o l e o p t i l e t i s s u e i n t h a t t h e

pH f o r maximal s t i m u l a t i o n o f m a iz e r o o t s e gm e n ts was found t o be

lower ( 3 . 5 - 4 . 0 ) t h a n t h a t o f c o l e o p t i l e t i s s u e ( 4 . 0 - 5 . 0 ) . I n b o t h

t i s s u e s t h e s t i m u l a t i o n o f g ro w th i s r a p i d ( w i t h i n 30 s e c ) . However ,

a c i d - i n d u c e d g ro w th i n r o o t s e g m e n t s was s h o r t e r i n d u r a t i o n

( c a . 1 - 2 h ) t h a n f o r o a t c o l e o p t i l e s ( c a . 6 h ) . Edwards and S c o t t

(1976) a l s o r e p o r t e d t h a t a c i d - s t i m u l a t e d g ro w th o f r o o t s was

r e - i n d u c i b l e . I f t h e pH o f t h e b a t h i n g medium were s h i f t e d from

pH 4 . 0 t o pH 7 . 0 , a c i d - i n d u c e d g r o w th c e a s e d w i t h i n a few m i n u t e s and

19

t h e g row th r a t e r e t u r n e d t o t h e p r e - t r e a t m e n t r a t e . A second

a c i d i f i c a t i o n o f t h e medium r e - i n i t i a t e d a c i d - i n d u c e d g r o w th . A

s i m i l a r r e - i n d u c i b l e a c i d s t i m u l a t i o n o f g ro w th was r e p o r t e d f o r pe a

r o o t s by Evans ( 1 9 7 6 ) . R a y le and C l e l a n d (1970 , 1972b) r e p o r t e d t h a t

a second r e s p o n s e t o a c i d i s n o t o b s e r v e d f o r o a t c o l e o p t i l e s .

The l i m i t e d i n f o r m a t i o n c o n c e r n i n g t h e c e l l w a l l l o o s e n i n g

mechanism and t h e c h a r a c t e r i s t i c s o f a c i d - i n d u c e d i n c r e a s e s i n c e l l

w a l l e x t e n s i b i l i t y o f r o o t t i s s u e i n d i c a t e d a need f o r i n v e s t i g a t i o n

o f t h e s e c h a r a c t e r i s t i c s .

M a t e r i a l s and Methods .

P l a n t M a t e r i a l .

Maize s e e d l i n g s ( Zea mays L . h y b r i d WF9 X MS38; C u s t o m a i z e ,

Momence, 1 1 1 . , USA) u s e d f o r t i s s u e e x t e n s i o n m easu rem en ts were

2 . 5 - 3 days o l d from the t im e o f p l a n t i n g . The m a iz e g r a i n s were

soaked i n d e m i n e r a l i z e d w a t e r w i t h a e r a t i o n f o r 24 h o u r s p r i o r t o

p l a n t i n g . The s e e d s were p l a n t e d i n rows on s t a n d a r d food s e r v i c e

t r a y s c o v e r e d w i t h w h i t e S c o t t p a p e r t o w e l s ( i n s t i t u t i o n a l g r a d e ) and

g e r m i n a t e d , i n a v e r t i c a l p o s i t i o n , i n a c o n t r o l l e d e n v i r o n m e n t g rowth

chamber (50% r e l a t i v e h u m i d i t y , 26 C day , 24 C n i g h t t e m p e r a t u r e ,

2l i g h t i n t e n s i t y 430 + / - 50 pE/M - s e c ) . E x p e r i m e n t s were c a r r i e d o u t2

u n d e r f l u o r e s c e n t room l i g h t i n g (175 + 25 pE/m - s e c ) a t room

20

t e m p e r a t u r e (25 + / - 3 C) . S e e d l i n g s u s e d f o r e l a s t i c / p l a s t i c

m ea su rem e n ts were f r o z e n i n l i q u i d n i t r o g e n f o r 5 - 1 0 m i n u t e s ,

r e t r i e v e d and a l l o w e d t o thaw t o room t e m p e r a t u r e . The f r e e z e / t h a w

c y c l e was r e p e a t e d 1 - 2 t i m e s .

S t r e s s / S t r a i n A p p a r a t u s .

Measurement o f e x t e n s i o n o f i n t a c t r o o t t i s s u e was m o n i t o r e d

u s i n g t h e a u xa nom e te r d e s c r i b e d p r e v i o u s l y by Evans (1976) as m o d i f i e d

by Kuzmanoff ( 1 9 7 9 ) . The t y p e o f chamber u s e d f o r t h e s e e x p e r i m e n t s

and f o r normal g ro w th e x p e r i m e n t s i s shown i n P l a t e I . A s t r i p o f

a luminum f o i l ( 1 . 5 x 0 . 5 cm, l e n g t h x w i d t h ) was a t t a c h e d t o t h e

a p i c a l 2 mm o f t h e f r o z e n / t h a w e d r o o t . A s m a l l n ich rom e w i r e loop

( c a . 5 mm i n d i a m e t e r ) was a t t a c h e d t o t h e o p p o s i t e end o f t h e

aluminum f o i l s t r i p . An a c r y l o n i t r i l e r a p i d c u r i n g g l u e ( E l m e r ' s

"Supe r G l u e " ) was u s e d t o a t t a c h t h e aluminum f o i l s t r i p t o b o t h t h e

r o o t t i p and t h e w i r e l o o p . W ate r l o s s f rom t h e f r o z e n / t h a w e d

s e e d l i n g and r o o t d u r i n g t h e a t t a c h m e n t o f t h e f o i l s t r i p was

m in im iz ed by c o v e r i n g t h e s e e d l i n g w i t h a m o i s t ( d e m i n e r a l i z e d w a t e r )

p a p e r t o w e l . The to w e l was d r a p e d o v e r t h e r o o t t o fo rm a s m a l l t e n t

t o a v o i d c o n t a c t w i t h t h e r o o t t i p . The g l u e was a l l o w e d t o c u r e f o r

15 min. A f t e r c u r i n g , t h e s e e d l i n g was mounted i n t h e g row th chamber

and a l l o w e d t o e q u i l i b r a t e f o r 30 min b e f o r e a t t a c h m e n t t o t h e

t r a n s d u c e r a s s e m b ly . T e n s i o n was a p p l i e d by a t t a c h i n g t h e r o o t t o t h e

P l a t e I .

Growth Chamber and A p p a r a t u s f o r Measurement o f P l a s t i c and E l a s t i cComponents o f Maize Root C e l l W al l M a t e r i a l .

1. P l e x i g l a s chamber u s e d f o r s t r e s s / s t r a i n m easu rem en ts and f o r g r o w th e x p e r i m e n t s .

2. A n g u l a r p o s i t i o n t r a n s d u c e r o r r o t a r y v a r i a b l e d i f f e r e n t i a l t r a n s f o r m e r .

3. T r a n s d u c e r arm.

4. Nichrome hook u s e d t o a t t a c h t h e t r a n s d u c e r arm t o t h e r o o t .

5 . E l e c t r o d e f o r m o n i t o r i n g t h e pH o f t h e b a t h i n g s o l u t i o n .

6. C a l i b r a t e d w e i g h t ( s ) f o r a p p l i c a t i o n o f t e n s i o n t o t h e r o o t .

7. M ic r o m e te r u s e d as a s a f e t y s t o p t o c o u n t e r b a l a n c e t h e a p p l i e d w e i g h t d u r i n g a d d i t i o n and r em o v a l o f t e n s i o n t o and from t h e r o o t .

21

P l a t e I .

23

t r a n s d u c e r arm by a s m a l l n i ch ro m e hook mounted a t t h e end o f t h e

t r a n s d u c e r arm ( P l a t e I I ) , t o form an eye and hook a s s e m b ly . The

w e i g h t o f t h e aluminum f o i l s t r i p and n i ch ro m e lo o p was a p p r o x i m a t e l y

50 mg. T e n s i o n was a p p l i e d t o t h e r o o t by l o a d i n g w e i g h t s o n t o t h e

t r a n s d u c e r arm ( P l a t e s I , I I I ) . The w e i g h t s were c a l i b r a t e d t o p r o d u c e

known f o r c e a t t h e hook a t t a c h e d t o t h e end o f t h e t r a n s d u c e r arm. A

100 mg w e i g h t was p r e s e n t a t a l l t im e s t o i n s u r e a r a p i d , u n i f o r m

t e n s i o n on t h e r o o t . The f o r c e s r e p o r t e d a r e t h e n e t f o r c e added t o

o r removed from t h e t r a n s d u c e r arm and do n o t i n c l u d e t h e a d d i t i o n a l

150 mg (50 mg f o r f o i l and lo o p , 100 mg w e i g h t on arm) which was

p r e s e n t t h r o u g h o u t t h e e x p e r i m e n t .

R e s u l t s and D i s c u s s i o n .

Measurement o f P l a s t i c / E l a s t i c R a t i o .

Two m a j o r component s o f t e n a s s o c i a t e d w i t h h e t e r o g e n o u s m a t e r i a l s ,

suc h as c e l l w a l l s o r t i s s u e s , a r e p l a s t i c i t y and e l a s t i c i t y . The

e l a s t i c component i s t h a t p o r t i o n o f t h e d e f o r m a t i o n , o r e x t e n s i o n ,

which i s r e v e r s i b l e . Upon r em o v a l o f t e n s i o n , p l a n t t i s s u e w i l l

r e t u r n , i n p a r t , t o i t s o r i g i n a l s i z e . I n t h e o r y , i f t h e f o r c e

a p p l i e d t o t h e t i s s u e i s s m a l l e r t h a n some c r i t i c a l l e v e l , t h e t i s s u e

w i l l r e t u r n t o i t s o r i g i n a l s h a p e ( i n p r a c t i c e , e v e n e l a s t i c i t y

e x h i b i t s h y s t e r e s i s a t t r i b u t e d t o v i s c o u s f l o w o r s h e a r i n g ) .

P l a t e I I .

A t t a c h m e n t o f Maize Roo t t o T r a n s d u c e r Arm.

The r o o t was a t t a c h e d t o t h e t r a n s d u c e r arm by means o f an a luminum f o i l s t r i p g l u e d t o t h e a p i c a l 1 - 2 mm o f t h e r o o t t i p . Theo p p o s i t e end o f t h e f o i l s t r i p was g l u e d t o a s m a l l n ich rom e w i r e loopw hich was t h e n a t t a c h e d t o t h e t r a n s d u c e r arm by means o f a n ich rom ehook mounted a t t h e end o f t h e arm.

1. Nichrome w i r e hook a t t a c h e d t o t h e t r a n s d u c e r arm which was u s e dt o a t t a c h t h e r o o t t o t h e arm.

2. Nichrome w i r e lo o p a t t a c h e d t o t h e aluminum f o i l s t r i p .

3. Aluminum f o i l s t r i p u s e d t o l i n k t h e r o o t t o t h e t r a n s d u c e r armhook . The f o i l s t r i p was shaped i n t o a c o l l a r and a t t a c h e d t o t h e r o o t t i p w i t h an a c r y l o n i t r i l e g l u e .

4. T r a n s d u c e r arm.

3. pH e l e c t r o d e .

24

P l a t e I I .

P l a t e I I I .

A p p l i c a t i o n o f T e n s i o n t o Maize R o o t s .

The w e i g h t s were f a b r i c a t e d from l e a d and sha v ed t o y i e l d t h e d e s i r e d f o r c e as m easu red a t t h e hook o f t h e t r a n s d u c e r arm. The p o s i t i o n on t h e t r a n s d u c e r arm ( a t t h e a r r o w , u p p e r l e f t c o r n e r ) f o r a p p l i c a t i o n o f t h e w e i g h t was n o t c h e d t o f a c i l i t a t e r e p r o d u c i b i l i t y .

26

27

P l a t e I I I .

Beyond t h e l i m i t o f e l a s t i c i t y , t h e t i s s u e i s p e r m a n e n t l y defo rmed

( e x t e n d e d ) and t h i s i r r e v e r s i b l e e x t e n s i o n i s a t t r i b u t e d t o t h e

p l a s t i c i t y o f t h e t i s s u e . E x t e n s i b i l i t y o f a t i s s u e i s d e f i n e d as t h e

s t r a i n / s t r e s s , whe re s t r a i n i s t h e d e f o r m a t i o n , o r e x t e n s i o n , and

s t r e s s i s t h e f o r c e / u n i t a r e a . S i n c e i n t a c t r o o t t i s s u e o f v e r y

n e a r l y t h e same age was u s e d i n a l l e x p e r i m e n t s , t h e m easu rem en t o f

s t r e s s i g n o r e d t h e c r o s s - s e c t i o n a l a r e a o f t h e t i s s u e . P r e l i m i n a r y

m easurem en ts i n d i c a t e d t h a t t h e d i a m e t e r o f t h e F/T m a iz e r o o t

e l o n g a t i o n z one , p r i o r t o a p p l i c a t i o n o f t e n s i o n , was b e tw e en 0 .9 5 and2

1 .18 mm. T h i s gave a c r o s s - s e c t i o n a l a r e a o f - 2 .9 8 - 3 . 7 mm and a

2s t r e s s o f a p p r o x i m a t e l y 6 0 . 5 + / - 6 . 5 g/cm f o r an a p p l i e d w e i g h t o f

2 g . The p l a s t i c / e l a s t i c r a t i o o f i n t a c t r o o t t i s s u e was e s t i m a t e d by

m e a s u r i n g t h e i r r e v e r s i b l e change i n l e n g t h b e f o r e and a f t e r a p p l y i n g

a c a l i b r a t e d w e i g h t t o t h e r o o t . A p p l i c a t i o n and r em o v a l o f t h e

t e n s i o n was c o n s i d e r e d t o be v i r t u a l l y i m m e d ia te . The i n i t i a l

e x t e n s i o n o b s e r v e d r e p r e s e n t s b o t h p l a s t i c and e l a s t i c e x t e n s i o n

( F i g . 1 ) . When t h e w e i g h t i s r emoved, t h e d e c r e a s e i n l e n g t h i s due

p r i m a r i l y t o t h e e l a s t i c component . E s t i m a t i o n o f t h e p l a s t i c i t y o f

t h e t i s s u e r e q u i r e s t h a t a f o r c e g r e a t e r t h a n t h e y i e l d s t r e s s be

a p p l i e d t o t h e t i s s u e . The y i e l d s t r e s s i s t h e f o r c e r e q u i r e d t o

i r r e v e r s i b l y de fo rm t h e t i s s u e , t h a t i s , t h e f o r c e n e c e s s a r y t o

overcome t h e r e s i s t a n c e t o p l a s t i c d e f o r m a t i o n . The y i e l d s t r e s s was

a p p r o x i m a t e d by a p p l y i n g i n c r e a s i n g f o r c e and m e a s u r i n g t h e change i n

l e n g t h o f t h e t i s s u e b e f o r e and a f t e r r em o v a l o f t h e w e i g h t .

F ig u r e 1.

Measurement o f E l a s t i c and P l a s t i c Components o f I n t a c t Frozen /Thawed Maize R o o t s .

A r e p r e s e n t a t i v e c u r v e i s p r e s e n t e d which d e m o n s t r a t e s t h e e l a s t i c i t y and p l a s t i c i t y o f m a iz e r o o t t i s s u e and t h e method o f a p p r o x i m a t i n g t h e s e p a r a m e t e r s . F o r c e s , as i n d i c a t e d , were a p p l i e d t o t h e r o o t and t h e f o r c e - i n d u c e d e x t e n s i o n m e a s u r e d . The p l a s t i c i t y was a p p r o x i m a t e d as t h a t f r a c t i o n o f t h e t o t a l e x t e n s i o n which was n o t r e v e r s i b l e a f t e r r em o v a l o f t h e w e i g h t . The p e r p e n d i c u l a r axes f o r e a c h p a i r o f e x t e n s i o n / r e l a x a t i o n c u r v e s i n d i c a t e t h e a p p r o x i m a t e d e l a s t i c and p l a s t i c f r a c t i o n s o f t h e to ta? . e x t e n s i o n . The p o r t i o n o f t o t a l e x t e n s i o n i n t h e f i r s t q u a d r a n t o f t h e axes r e p r e s e n t s t h e e l a s t i c component . The s e cond q u a d r a n t i l l u s t r a t e s t h e p l a s t i c com ponent . M easurements were r e p e a t e d a t l e a s t f i v e t im e s f o r ea ch w e i g h t .

29

L e n g t h (mm)

ro

31

F r o z e n / t h a w e d m aize r o o t s e x h i b i t e d a p l a s t i c component above 400 mg

( n e t ) . I r r e v e r s i b l e e x t e n s i o n o f t h e t i s s u e c o n t i n u e d i n a r e l a t i v e l y

l i n e a r f a s h i o n o v e r t h e r a n g e o f 1 - 2 g o f a p p l i e d w e i g h t ( F i g . 2 ) .

The a b s o l u t e m ag n i tu d e o f b o t h t h e e l a s t i c and p l a s t i c components

i n c r e a s e d w i t h i n c r e a s i n g t e n s i o n . At 250 mg t e n s i o n t h e r e a p p e a r e d

t o be a s h a r p i n c r e a s e i n t h e p l a s t i c i t y which was l a r g e r t h a n t h e

p l a s t i c i t y o b s e r v e d a t e i t h e r 0 . 5 o r 1 . 0 g o f t e n s i o n . The anomolous

i n c r e a s e i n p l a s t i c i t y f o r 250 mg o f w e i g h t , a l t h o u g h v a r i a b l e , was

r e p e a t a b l e and s u g g e s t e d e i t h e r t h a t more t h a n one l e v e l o f c r i t i c a l

s t r e s s f o r r o o t c e l l w a l l m a t e r i a l o r t h a t u n i a x i a l t e n s i o n upon t h e

t i s s u e p r o d u c e s t e n s i l e s h e a r i n g . A p p r o x i m a t e l y 50 % o f t h e t o t a l

e x t e n s i o n was i r r e v e r s i b l e f o r 2 g o f a p p l i e d s t r e s s ( T a b le 1 ) .

A c i d i f i c a t i o n o f t h e b a t h i n g s o l u t i o n t o pH 4 . 0 r e s u l t e d i n a d d i t i o n a l

e x t e n s i o n r e s u l t i n g i n n e a r l y a two f o l d i n c r e a s e i n t h e e x t e n s i b i l i t y

( F i g . 3 ) . No s i g n i f i c a n t change i n e l a s t i c i t y was o b s e r v e d . Low pH,

i n t h e p r e s e n c e o f t e n s i o n , i n d u c e d f u r t h e r i r r e v e r s i b l e e x t e n s i o n o f

t h e c e l l w a l l m a t e r i a l . T r e a t m e n t w i t h a c i d i n t h e a b s e n c e o f t e n s i o n

d i d n o t i n d u c e s i g n i f i c a n t o r r e p r o d u c i b l e e x t e n s i o n when t h e t i s s u e

was r e t u r n e d t o pH 6 . 5 w i t h a p p l i e d t e n s i o n . T r e a t m e n t o f F /T t i s s u e

w i t h 10 mM EDTA ( i n ImM MOPS, pH 6 . 5 ) , w h i l e u n d e r t e n s i o n , r e s u l t e d

i n a l a r g e , r a p i d i n c r e a s e i n l e n g t h . The i n c r e a s e i n l e n g t h r e s u l t e d

F ig u r e 2.

D e t e r m i n a t i o n o f t h e Y i e l d S t r e s s o f I n t a c t . F /T Maize R o o t s .

E l a s t i c i t y and p l a s t i c i t y were m easu red as i n F i g u r e 1. The p l o t t e d v a l u e s a r e t h e a v e r a g e o f f i v e m ea su rem e n ts w i t h + / - 0 . 5 s t a n d a r d d e v i a t i o n s . The p l a s t i c i t y o f t h e t i s s u e becomes a p p a r e n t f o r t e n s i o n g r e a t e r t h a n 0 . 4 g i n d i c a t i n g t h a t t h e y i e l d s t r e s s i s b e tw e e n 0 . 4 and 0 . 5 g o f a p p l i e d u n i a x i a l t e n s i o n .

32

Figure 2

Plasticity/ Elasticity

o

o09

0>

N)b

ee

T able 1.

C a l c u l a t e d E l a s t i c and P l a s t i c F r a c t i o n s o f S t r e s s - I n d u c e d E x t e n s i o n o f Maize R o o t s .

V a lue s f o r e l a s t i c i t y and p l a s t i c i t y were m ea su red f i v e t i m e s u s i n g t h e method shown i n F i g u r e 1. The e l a s t i c and p l a s t i c f r a c t i o n s a r e p r e s e n t e d as f r a c t i o n s o f t h e t o t a l l e n g t h . The t o t a l l e n g t h i s t h e sum o f p l a s t i c and e l a s t i c e x t e n s i o n .

34

35

T a b l e 1.

T e n s i o nA p p l i e d

T o t a le x t e n s i o n

E l a s t i cf r a c t i o n

P l a s t i cf r a c t i o n

P/E P/T

0 .1 0 g 0 .0 3 8 0 .0 3 8 0 0 0

0 .2 5 g 0 .0 6 7 0 .0 5 0 0 .0 1 7 0 .3 4 0 .2 5

0 .5 0 g 0 .0 8 9 0 .0 8 4 0 .0 0 5 0 .05 0 .0 6

1 .0 g 0 .1 95 0 .1 6 0 0 .0 3 5 0 .2 2 0 .1 8

1 .5 g 0 .2 3 8 0 .1 4 0 0 .0 9 8 0 .6 4 0 . 3 9

2 . 0 g 0 .2 28 0 .1 1 0 0 .1 2 0 1 .07 0 . 5 2

34

F ig u r e 3.

A c i d - and EDTA-Induced I n c r e a s e i n Maize Root E x t e n s i b i l i t y .

A r e p r e s e n t a t i v e e x p e r i m e n t d e p i c t i n g t h e i n c r e a s e i n e x t e n s i b i l i t y r e s u l t i n g from a c i d i f i c a t i o n o f F /T m aize r o o t s i n t h e p r e s e n c e o f 2 g o f t e n s i o n . A c i d i f i c a t i o n t o pH 4 . 0 r e s u l t e d i n e x t e n s i o n which be g a n a lm o s t i m m e d i a t e l y . The a c i d - i n d u c e d e x t e n s i o n was p r i m a r i l y an e f f e c t upon t h e p l a s t i c component , s i n c e rem ova l o f t e n s i o n ( s e c o n d a r r o w ) r e s u l t e d i n c o n t r a c t i o n c o m p a rab le t o t h a t o f c o n t r o l s . * T h i s s u g g e s t s t h a t t h e r e was no i n c r e a s e i n e l a s t i c i t y .The d i v a l e n t c a t i o n c h e l a t o r EDTA a l s o i n d u c e s e x t e n s i o n which was s i m i l a r t o t h a t i n d u c e d by a c i d . The EDTA e f f e c t was o b s e r v e d even

I a f t e r a c i d - i n d u c e d e x t e n s i o n was e x h a u s t e d .

36

J 1 I I0 40

I I I__80 120

Minutes

F ig u r e 3.

10 mM EDTA

J I I I L160 200

_I_220

p r i m a r i l y f rom an i n c r e a s e i n t h e p l a s t i c e x t e n s i b i l i t y which

i n c r e a s e d a s much as t h r e e f o l d i n r e s p o n s e t o t r e a t m e n t w i t h EDTA

w h i l e u n d e r t e n s i o n ( F i g . 4 ) . EDTA-induced e x t e n s i o n was v a r i a b l e ,

b u t i n a l l c a s e s t h e i n c r e a s e i n e x t e n s i b i l i t y i n d u c e d by 10 mM EDTA

was as l a r g e as t h e i n c r e a s e r e s u l t i n g from t r e a t m e n t o f t h e t i s s u e

w i t h a c i d i c b u f f e r (pH 4 . 0 ) . However, t h e a b i l i t y o f EDTA t o i n c r e a s e

e x t e n s i b i l i t y a p p e a r s t o be r e d u c e d by p r e - t r e a t m e n t w i t h a c i d .

A l t h o u g h e x t e n s i o n i n d u c e d by EDTA was s i m i l a r t o t h e p r e - t r e a t m e n t

a c i d - i n d u c e d e x t e n s i o n , t h e EDTA-induced e x t e n s i o n a f t e r a c i d

t r e a t m e n t ( F i g . 3) i s s m a l l e r t h a n t h a t o b s e r v e d f o l l o w i n g EDTA

t r e a t m e n t a l o n e ( F i g . 4 ) . The i n c r e a s e i n l e n g t h o b s e r v e d f o l l o w i n g

t r e a t m e n t w i t h 10 mM EDTA was i n d u c i b l e e ven a f t e r t r e a t m e n t o f t h e

t i s s u e w i t h a c i d pH ( F i g . 3 ) . S i m i l a r r e s u l t s were o b t a i n e d when EGTA

was s u b s t i t u t e d f o r EDTA ( d a t a n o t shown) . However , t h e i n c r e a s e i n

l e n g t h r e s u l t i n g from a l o w e r i n g o f t h e pH was a l m o s t c o m p l e t e l y

a b o l i s h e d i f a c i d i f i c a t i o n was p r e c e d e d by EDTA t r e a t m e n t ( F i g . 5 ) .

The s l i g h t a c i d - i n d u c i b l e e x t e n s i o n which r e m a in e d f o l l o w i n g EDTA

t r e a t m e n t , was n o t a b o l i s h e d by h i g h e r c o n c e n t r a t i o n s o f EDTA. T h i s

s u g g e s t s t h a t t h e normal a c i d - i n d u c e d e x t e n s i o n c o n s i s t s o f two p a r t s ,

o n l y one o f which i s EDTA s e n s i t i v e . The EDTA i n s e n s i t i v e component

may r e p r e s e n t a d i r e c t e f f e c t o f low pH e i t h e r upon c a t i o n

d i s p l a c e m e n t o r h y d r o g e n bond d i s r u p t i o n w i t h i n t h e c e l l w a l l . T h i s

component o f t h e a c i d - i n d u c e d c e l l w a l l l o o s e n i n g a p p e a r s s i m i l a r t o

F ig u r e 4 .

E s t i m a t i o n o f P l a s t i c and E l a s i t c Components A f t e r T r e a t m e n t o f F/T T i s s u e w i t h EDTA.

EDTA can i n d u c e a l a r g e i n c r e a s e i n t h e p l a s t i c i t y o f t h e t i s s u e w i t h o u t a l t e r i n g t h e e l a s t i c component . I n most c a s e s , t h e EDTA-induced e x t e n s i o n was n o t as l a r g e as shown and was more c o m p a r a b le t o a c i d - i n d u c e d e x t e n s i o n ( F i g s . 3 , 5 ) . T h i s e x p e r i m e n t r e p r e s e n t s t h e u p p e r l i m i t o f EDTA enhanced c e l l w a l l l o o s e n i n g .

39

mm

1.0

0 8

06

04i

02 ii

ii

< —10mM EOT A

J 1 I I LI I i I3 0 6 0 9 0 1 5 0Minutes

o

F ig u r e 4 .

F ig u r e 5 .

E f f e c t o f EDTA Upon A c i d - I n d u c e d E x t e n s i o n o f F /T Maize R o o t s .

A r e p r e s e n t a t i v e c u r v e showing t h e a c t i o n o f EDTA upon c e l l w a l l l o o s e n i n g o f f r o z e n / t h a w e d r o o t s w i t h a p p l i e d t e n s i o n . A p p l i c a t i o n o f EDTA i n t h e p r e s e n c e o f 2 g t e n s i o n r e s u l t e d i n a l a r g e i n c r e a s e i n e x t e n s i o n which was p r i m a r i l y an i n c r e a s e i n t h e c e l l w a l l p l a s t i c i t y . A t t h e s e c o n d a r r o w t h e t e n s i o n was removed and t h e t i s s u e was a l l o w e d t o r e l a x . R e - a p p l i c a t i o n o f t h e w e i g h t e x t e n d e d t h e t i s s u e and r e v e a l e d t h e amount o f r e v e r s i b l e , e l a s t i c e x t e n s i o n p r e s e n t .T r e a t m e n t w i t h a c i d i c b u f f e r r e s u l t e d i n o n l y a s m a l l i n c r e a s e i n e x t e n s i o n . P r e - t r e a t m e n t w i t h EDTA a b o l i s h e d most o f t h e a c i d - i n d u c e d e x t e n s i o n . A s m a l l a c i d e f f e c t r e m a in e d a f t e r EDTA t r e a t m e n t ( f o u r t h a r r o w ) .

41

08

06

£ 0.4

10mMEDTA02

x x x XX X0 4 0 8 0 120

Minutes

F igu re 5 .

r

t♦2g

■ ■____ ■ ■ ■_____L_160 2 0 0 220

N>

43

t h e a c i d e f f e c t o b s e r v e d f o r a l g a l c e l l w a l l s ( T e p f e r and

C l e l a n d , 1979 ) . A d d i t i o n o f EDTA a t h i g h e r c o n c e n t r a t i o n s t o

f r o z e n / t h a w e d r o o t s in d u c e d f u r t h e r e x t e n s i o n ( F i g . 6 ) . A p o r t i o n o f

t h i s e x t e n s i o n r e s u l t s f rom t h e h i g h o s m o t i c p o t e n t i a l o f t h e EDTA

s o l u t i o n which a p p e a r s t o w i t h d r a w w a t e r f rom t h e r o o t r e s u l t i n g i n

some l a t e r a l c o l l a p s e a l l o w i n g i n c r e a s e d e x t e n s i o n u n d e r s t r e s s .

A p p l i c a t i o n o f EDTA t o i n t a c t , g row ing t i s s u e u n d e r s t r e s s ,

r e s u l t e d i n a r a p i d i n c r e a s e i n l e n g t h which c l o s e l y a p p r o x i m a t e s t h e

r e s p o n s e o f r o o t t i s s u e t o d e c r e a s e d e x t e r n a l pH ( F i g . 7 ) . I n c r e a s i n g

t h e c o n c e n t r a t i o n o f EDTA f rom 10 t o 100 mM r e s u l t e d i n b o t h g r e a t e r

and more r a p i d e x t e n s i o n o f t h e t i s s u e . However, t h e d u r a t i o n o f t h e

r e s p o n s e o f g row ing t i s s u e ( u n d e r t e n s i o n ) t o EDTA was s h o r t e r t h a n

t h a t o f t h e r e s p o n s e t o a c i d i f i c a t i o n o f t h e e x t e r n a l medium ( F i g . 7 ) .

High c o n c e n t r a t i o n s o f EDTA, a l t h o u g h i n i t i a l l y s t i m u l a t o r y , q u i c k l y

i n h i b i t e d g r o w t h . Such i n h i b i t i o n a p p e a r s t o r e s u l t f rom t h e o s m o t i c

e f f e c t o f EDTA and t h e s t r o n g c h e l a t i n g a c t i o n o f t h e compound. Over

t h e c o n c e n t r a t i o n r a n g e o f 10 - 100 mM, t h e e f f e c t o f EDTA on

e x t e n s i o n was n o t o b s e r v e d f o r g row ing r o o t s w i t h o u t a p p l i e d t e n s i o n .

P r o t e i n d e n a t u r a t i o n o f F /T t i s s u e u s i n g t h e c h a o t r o p i c a g e n t ,

u r e a (8 M), or b o i l i n g t h e t i s s u e i n m e t h a n o l (15 m i n u t e s ) a l s o

a b o l i s h e d a c i d - i n d u c e d e x t e n s i o n . The e x t e n s i b i l i t y o f t h e t i s s u e

i n c r e a s e d o n l y s l i g h t l y i n r e s p o n s e t o a c i d i c s o l u t i o n s (pH 4 . 0 ) . The

amount o f e x t e n s i o n in d u c e d by a c i d t r e a t m e n t was s i m i l a r t o t h e s m a l l

a c i d - i n d u c e d e x t e n s i o n f o l l o w i n g EDTA-induced e x t e n s i o n ( F i g . 8 ) .

F ig u r e 6.

E f f e c t o f I n c r e a s i n g C o n c e n t r a t i o n s o f EDTA Upon C e l l W a l l L o o s e n in g o f F roz en /T ha w e d Maize R o o t s .

The m a g n i tu d e o f EDTA-induced e x t e n s i o n i n c r e a s e d as t h e c o n c e n t r a t i o n o f EDTA was i n c r e a s e d . A s m a l l p o r t i o n o f t h e e x t e n s i o n a p p e a r s t o r e s u l t f rom t h e d e h y d r a t i o n o f t h e c e l l w a l l m a t e r i a l . I f t h e 100 mM EDTA s o l u t i o n were removed and t h e t i s s u e were r e t u r n e d to t h e p r e - t r e a t m e n t s o l u t i o n , a s m a l l amount o f s h r i n k a g e o c c u r r e d w h i l e t h e t i s s u e was s t i l l u n d e r t e n s i o n .

44

0 20 4 0 6 0 80

Minutes

F igure 6 .

1/2 M eyer's

5 0 mM EDTA lOOmM EDTA

120100 140 160 180

Ui

F ig u r e 7 .

EDTA M im icry o f A c i d - I n d u c e d Growth i n L i v in g ^ I n t a c t Maize R o o t s .

A p p l i c a t i o n o f EDTA t o g row ing r o o t s i n t h e p r e s e n c e o f a s m a l l amount o f t e n s i o n ( 0 . 5 g) r e s u l t e d i n a r a p i d i n c r e a s e i n g row th r a t e . EDTA c o n c e n t r a t i o n s above 25 mM a p p e a r e d t o be t o x i c . A l th o u g h th e g r o w th i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f EDTA, t h e d u r a t i o n o f t h e s t i m u l a t i o n became s h o r t e r . By c o m p a r i s o n t o a c i d i n d u ce d g r o w t h , EDTA s t i m u l a t i o n was s h o r t e r i n d u r a t i o n even a t 10 mM.

46

Leng

th

(mm

)

47

a2mM EDTA

2B 102550

1002.4

20

1.6

1.2

OB

0.4

0.4

- 0.8

12080 1600 4040M i n u t e s

F ig u r e 7 .

F ig u r e 8 .

Loss o f A c i d - I n d u c e d E x t e n s i o n A f t e r T r e a t m e n t w i t h U r e a .

A f t e r no rm a l t e n s i o n - i n d u c e d e x t e n s i o n o f f r o z e n / t h a w e d i n t a c t m a i z e r o o t t i s s u e , t h e t e n s i o n was removed and t h e t i s s u e was t r e a t e d w i t h 8 M u r e a a d j u s t e d t o pH 6 . 5 f o r 15 m i n u t e s . The t i s s u e was g e n t l y r i n s e d w i t h d i s t i l l e d w a t e r and t e n s i o n r e a p p l i e d t o t h e r o o t . E x t e n s i o n r e s u l t i n g from u r e a t r e a t m e n t was r a p i d . A d j u s tm e n t o f t h e b a t h i n g medium t o pH 4 . 0 f o l l o w i n g t r e a t m e n t w i t h u r e a in d u c e d a s m a l l e x t e n s i o n s i m i l a r i n s i z e and d u r a t i o n t o t h e a c i d - i n d u c e d e x t e n s i o n o b s e r v e d a f t e r EDTA t r e a t m e n t . The m a j o r i t y o f t h e a c i d - i n d u c e d e x t e n s i o n was a b o l i s h e d . However , EDTA a t 50 mM in d u c e d a l a r g e , r a p i d e x t e n s i o n . P r o p o r t i o n a l l y s m a l l e r e x t e n s i o n was in d u c e d by lo w e r c o n c e n t r a t i o n s o f EDTA.

48

Leng

th

(mm

)

12

10

as

ae

5 0 mM EDTA

02 8M UREA

< - 2g

20 4 0 110 130 150 170 190 210Minutes

-P-vo

F igu re 8 .

I n summary, f r o z e n / t h a w e d m aize r o o t t i s s u e has a y i e l d s t r e s s

n e a r 0 . 4 g and e x h i b i t e d i n c r e a s e d c e l l w a l l l o o s e n i n g ( e x t e n s i b i l i t y )

when t r e a t e d w i t h e i t h e r a c i d o r EDTA i n t h e p r e s e n c e o f a p p l i e d

t e n s i o n . I n c o n t r a s t t o o a t c o l e o p t i l e F /T t i s s u e , t h e EDTA-induced

i n c r e a s e i n e x t e n s i b i l i t y o f F/T m aize r o o t t i s s u e was l a r g e , r a p i d

and c l o s e l y a p p ro x i m a t e d t h e e x t e n s i o n r e s p o n s e i n d u ce d by a c i d

t r e a t m e n t . I n t h i s r e s p e c t , m a ize r o o t c e l l w a l l m a t e r i a l more

c l o s e l y r e s e m b l e s V a l o n i a . However , p r o t e i n d e n a t u r i n g t r e a t m e n t o f

V a l o n i a w a l l m a t e r i a l a b o l i s h e s n e i t h e r a c i d n o r EDTA s t i m u l a t e d

e x t e n s i o n . A c i d - i n d u c e d e x t e n s i o n i n o a t c o l e o p t i l e and m aize r o o t

F/T t i s s u e i s l o s t a f t e r s u c h d e n a t u r i n g t r e a t m e n t s . T e p f e r and

C l e l a n d (1979) c o n c lu d e d t h a t t h e mechani sm o f CWL i n o a t c o l e o p t i l e s

was p r o b a b l y e n z y m a t i c w h i l e t h a t o f V a l o n i a was d e p e n d e n t p r i m a r i l y

upon a c h e m i c a l d i s r u p t i o n o f c e l l w a l l bonds by a c i d . The l o s s o f

a c i d - i n d u c e d e x t e n s i o n a f t e r d e n a t u r a t i o n o f m a ize t i s s u e s u g g e s t s an

e n z y m a t i c CWL mechanism. The l a r g e EDTA-induced e x t e n s i o n e x h i b i t e d

by m a iz e r o o t t i s s u e s u g g e s t s a p r i m a r y CWL mechanism i n v o l v i n g c a t i o n

d i s p l a c e m e n t , s i m i l a r t o t h a t o c c u r r i n g i n V a l o n i a . However, a w a l l

l o o s e n i n g mechanism i n m a iz e r o o t t i s s u e b a s e d s t r i c t l y on

p h y s i c o - c h e m i c a l d i s p l a c e m e n t o f c a l c i u m i s i n c o n s i s t e n t w i t h t h e

o b s e r v a t i o n t h a t enzyme d e n a t u r i n g t r e a t m e n t s r e n d e r m aize r o o t t i s s u e

i n s e n s i t i v e t o a c i d pH. T h i s i n d i c a t e s t h a t w a l l l o o s e n i n g i n v o l v e s

51

b o t h enzyme a c t i o n and c a l c i u m d i s p l a c e m e n t , p e r h a p s an

e n z ym e-m ed ia ted d i s p l a c e m e n t o f c a l c i u m f rom t h e c e l l w a l l . I n

c o n t r a s t t o V a l o n i a , t h e i n a b i l i t y o f a c i d , i n t h e a b s e n c e o f t e n s i o n ,

t o i n c r e a s e m aize CWL s u g g e s t s t h a t d i v a l e n t c a t i o n s i n m aize r o o t

c e l l w a l l s do n o t f u n c t i o n p r i m a r i l y as c r o s s - l i n k i n g com ponen ts .

Maize r o o t d i f f e r s f rom c o l e o p t i l e c e l l w a l l m a t e r i a l i n t h a t a s m a l l

p o r t i o n o f t h e a c i d - i n d u c e d e x t e n s i o n was n o t a b o l i s h e d by e i t h e r EDTA

or d e n a t u r a t i o n p r e - t r e a t m e n t . The p r e s e n c e o f t h i s r e s i d u a l

a c i d - i n d u c i b l e e x t e n s i o n s u g g e s t s t h a t m a iz e r o o t s p o s s e s s b o t h an

e n z y m a t i c and a p h y s i c o - c h e m i c a l CWL mechan ism, w i t h t h e e n z y m a t i c

p r o c e s s b e i n g t h e m a jo r mode o f CWL. S i n c e c a l c i u m may p l a y an

i m p o r t a n t r o l e i n t h e r e g u l a t i o n o f m a iz e r o o t c e l l w a l l l o o s e n i n g ,

t h e e f f e c t o f c a l c i u m upon m a iz e r o o t g row th was exam ined , as

d e s c r i b e d i n t h e f o l l o w i n g c h a p t e r .

C h a p t e r I I . Measurement o f Root Growth a s A f f e c t e d by C a lc iu m and

I n h i b i t o r s o f C a l m o d u l i n .


Growth in p l a n t s i s d e p e n d e n t upon t h e b i o c h e m i c a l l o o s e n i n g o f

t h e c e l l w a l l ( w i t h c o n c o m i t a n t a d d i t i o n o f new w a l l m a t e r i a l ) and t h e

m a i n t e n a n c e o f t u r g o r . The p r e s s u r e w i t h i n t h e t u r g i d p l a n t c e l l

p l a c e s m u l t i a x i a l t e n s i o n upon t h e c e l l w a l l . A l th o u g h t h e mechanism

o f c e l l w a l l l o o s e n i n g i s b i o c h e m i c a l , t h e p r o c e s s a l s o r e q u i r e s

c o n t i n u e d t e n s i o n . A u x i n - s t i m u l a t e d g ro w th a p p e a r s t o r e g u l a t e , i n

p a r t , b o t h c e l l e x t e n s i b i l i t y and o s m o t i c p o t e n t i a l . Masuda (1978)

and C l e l a n d ( 1 9 6 7 a ) d e m o n s t r a t e d t h a t a u x i n t r e a t m e n t o f p e a s tem

i n t e r n o d e and o a t c o l e o p t i l e t i s s u e , p r i o r t o d i s r u p t i o n o f t h e

p r o t o p l a s t ( f r e e z e / t h a w ) , r e s u l t e d i n an i n c r e a s e d e x t e n s i b i l i t y o f

t h e c e l l w a l l m a t e r i a l . Auxin s t i m u l a t e d g ro w th i n p e a s tem ( C o a r t n e y

and M orre , 19 8 0 ) , and o a t c o l e o p t i l e ( C l e l a n d , 1967b; R a y le and

C l e l a n d , 1972b) t i s s u e c a n be a b r u p t l y h a l t e d by t r e a t m e n t w i t h

s o l u t i o n o f r e l a t i v e l y low o s m o t i c p o t e n t i a l ( 0 . 1 - 0 . 2 5 M m a n n i t o l ) .

I n c o n t r a s t t o t h e s e o b s e r v a t i o n s , t h e i n h i b i t i o n o f r o o t g rowth

r e q u i r e s c o n s i d e r a b l y h i g h e r c o n c e n t r a t i o n s o f o smot icum ( g r e a t e r t h a n

0 . 5 M m a n n i t o l ) (Kuzmanoff , 1979, 1 9 8 1 ) .

52

T r e a t m e n t o f c e l l w a l l m a t e r i a l ( f r o z e n / t h a w e d t i s s u e ) w i t h

a c i d i c s o l u t i o n s w i l l a l s o r e s u l t i n c e l l w a l l l o o s e n i n g . However ,

a c i d i c s o l u t i o n s o n l y i n d u c e c e l l w a l l l o o s e n i n g when a p p l i e d t o

t i s s u e u n d e r t e n s i o n ( e i t h e r by n a t u r a l t u r g o r o r by a p p l i c a t i o n o f a

w e i g h t ) . T h i s s u g g e s t s t h a t a c i d may mimic a u x i n - i n d u c e d c e l l w a l l

l o o s e n i n g by a c t i n g d i r e c t l y upon t h e c e l l w a l l . T h i s i s c o n s i s t e n t

w i t h t h e h y p o t h e s i s t h a t H+ f u n c t i o n as s e c o n d a r y m e s s e n g e r s f o r

a u x i n . The a c t i o n o f a u x i n i n c e l l w a l l l o o s e n i n g a p p e a r s t o be

d e p e n d e n t upon a b i o c h e m i c a l mechanism s i n c e t r e a t m e n t o f

f r o z e n / t h a w e d o a t c o l e o p t i l e t i s s u e w i t h a u x i n , e v e n i n t h e p r e s e n c e

o f t e n s i o n , does n o t i n c r e a s e t h e e x t e n s i b i l i t y ( c e l l w a l l l o o s e n i n g )

o f t h e c e l l w a l l .

The d e pe ndenc e upon m e t a b o l i s m o f t h e a u x i n - i n d u c e d r e s p o n s e has

been d e m o n s t r a t e d t h r o u g h t h e u s e o f r e s p i r a t o r y i n h i b i t o r s s u c h as

i o d o a c e t i m i d e , KCN, ( C l e l a n d , 1967a) and oxygen d e p r i v a t i o n ( R a y le and

C l e l a n d , 1972b ) . T h e r e i s c o n s i d e r a b l e e v i d e n c e t h a t a u x i n - i n d u c e d

g row th i s b i p h a s i c ( V a n d e rh o e f and D u te , 1 9 8 1 ) . The two p h a s e s

i n c l u d e an e a r l y component , a t t r i b u t a b l e t o a u x i n - i n d u c e d w a l l

a c i d i f i c a t i o n , and l a s t i n g a b o u t 1 h o u r , and a s e c o n d l o n g e r - t e r m

component r e s p o n s i b l e f o r s u s t a i n e d , s u b s t a n t i a l a u x i n - i n d u c e d g row th

ove r p e r i o d s o f s e v e r a l t o many h o u r s . . D u r ing t h i s " se c o n d p h a s e " ,

c o n t i n u e d c e l l w a l l l o o s e n i n g must o c c u r . V a n d e r h o e f and Dute (1981)

s p e c u l a t e d t h a t t h e s e c o n d p h a s e r e q u i r e s a u x i n - s t i m u l a t e d s y n t h e s i s

o f c e l l w a l l p o l y m e r s , t o m a i n t a i n t h e s u p p l y o f s u b s t r a t e f o r t h e

w a l l - l o o s e n i n g ( low pH) r e a c t i o n . A u x i n - s t i m u l a t e d g r o w th h a s b e e n

shown t o be d e p e n d e n t upon c o n t i n u e d p r o t e i n s y n t h e s i s . P r e - t r e a t m e n t

w i t h c y c l o h e x i m i d e f o r 30 min c o m p l e t e l y i n h i b i t s b o t h t h e f i r s t and

second p h a s e o f t h e no rm a l a u x i n - i n d u c e d g ro w th r e s p o n s e (R a y le and

C l e l a n d , 1972b) .

Bonds be tw e en t h e m o l e c u l a r components o f t h e c e l l w a l l must be

b r o k e n . S i n c e a c i d i c b u f f e r s can mimic a u x i n s t i m u l a t i o n o f g row th

(R a y le and C l e l a n d , 1972a; T e p f e r and C l e l a n d , 1979) and s i n c e H+

s e c r e t i o n , a p p a r e n t l y f rom t h e c y t o p l a s m i n t o t h e c e l l w a l l , p r e c e d e s

a u x i n s t i m u l a t e d e l o n g a t i o n ( J a c o b s and Ray, 1 9 7 6 ) , i t seems p r o b a b l e

t h a t a u x i n - i n d u c e d w a l l a c i d i f i c a t i o n m e d i a t e s t h e w a l l l o o s e n i n g

p r o c e s s e s s e n t i a l f o r g r o w t h . T h i s would s u g g e s t t h a t t h e i n i t i a l

i n d u c t i o n o f H+ s e c r e t i o n by a u x i n may be n e c e s s a r y t o a c t i v a t e o r

s t i m u l a t e an e x o - o r e n d o - g l y c o s i d a s e , p r e s e n t w i t h i n t h e c e l l w a l l ,

which e x h i b i t s o p t i m a l (m aximal) a c t i v i t y a t low pH. Heyn (1981) has

a rg u e d t h a t d e x t r a n a s e , which has a pH opt imum n e a r 4 , i s a m a jo r

component o f c e l l w a l l l o o s e n i n g . However , t h i s d e x t r a n a s e h a s n o t

b e e n shown t o be s p e c i f i c a l l y s t i m u l a t e d by a u x i n . Nev ins ( 1 9 7 5 a , b )

has s u g g e s t e d t h a t a B - e x o g l u c a n a s e c a n mimic no rm a l a u x i n - i n d u c e d c e l l

w a l l l o o s e n i n g . A g a in , a s p e c i f i c i n c r e a s e i n enzyme a c t i v i t y i n

r e s p o n s e t o a u x i n h a s n o t b e e n d e m o n s t r a t e d .

55

A l th o u g h t h e b i o c h e m i c a l b a s i s o f c e l l w a l l l o o s e n i n g has n o t

been d e t e r m i n e d , J a c o b s and Ray (1975) have shown t h a t a w a t e r s o l u b l e

x y l o g l u c a n i s r e l e a s e d d u r i n g b o t h a u x i n - and a c i d - i n d u c e d s t i m u l a t i o n

o f p e a s tem e l o n g a t i o n . T h i s po lymer a p p e a r s t o be r e l e a s e d from a

h e r a i c e l l u l o s i c f r a c t i o n o f t h e c e l l w a l l ( L a b a v i t c h and Ray, 1 9 7 4 a ) ,

and may be t h e d e g r a d a t i o n p r o d u c t r e s u l t i n g f rom c e l l w a l l l o o s e n i n g

( L a b a v i t c h and Ray, 1 9 7 4 b ) . The x y l o g l u c a n i s s y n t h e s i z e d by t h e

c o o p e r a t i v e a c t i o n o f a 1 3 -1 ,4 -g lu c an s y n t h a s e and a UDP-xy lose x y l o s y l

t r a n s f e r a s e , b o t h o f w h ich have b e e n l o c a l i z e d i n G o l g i v e s i c l e s (Ray,

1980 ) . The d e pe ndenc e o f a u x i n - i n d u c e d g rowth upon p r o t e i n s y n t h e s i s

and t h e p r o p o s a l t h a t H+ f u n c t i o n a s s e c o n d a r y m e s s e n g e r s f o r a u x i n

s u g g e s t t h a t w a l l - b o u n d x y l o g l u c a n may be one o f t h e s u b s t r a t e s

n e c e s s a r y f o r t h e c e l l w a l l l o o s e n i n g r e a c t i o n . Ray (1980) showed

t h a t a u x i n t r e a t m e n t o f p e a t i s s u e i n c r e a s e d t h e a c t i v i t y o f t h e G o l g i

membrane B - g l u c a n s y n t h a s e and x y l o s y l t r a n s f e r a s e r e s p o n s i b l e f o r

x y l o g l u c a n b i o s y n t h e s i s . T h i s e f f e c t c o u l d be t h e b a s i s , t h e r e f o r e ,

f o r t h e s u s t a i n e d , s e c o n d p h a s e , a u x i n g ro w th r e s p o n s e .

R e c e n t l y , T h e o l o g i s and Ray (1982) and Z u r f l u h and G u i l f o y l e

( 1 9 8 2 a , b ) h a v e shown, u s i n g v i t r o t r a n s l a t i o n , t h a t s e v e r a l mRNAs

i n c r e a s e d r a p i d l y d u r i n g a u x i n t r e a t m e n t . F i v e new mRNA s p e c i e s

i n c r e a s e d s u b s t a n t i a l l y w i t h i n 2 h o u r s . Of t h e s e , 2 - 3 showed a

s i g n i f i c a n t i n c r e a s e w i t h i n 20 m i n u t e s . As a r e s u l t o f t h e s e mRNA

i n c r e a s e s , a u x i n may s t i m u l a t e de novo s y n t h e s i s o f s p e c i f i c

p o l y p e p t i d e s . The B - l , 4 - g l u c a n s y n t h a s e d e s c r i b e d by Ray (1973)

56

i s one o f t h e few known enzymes t h a t i n c r e a s e s i n a c t i v i t y w i t h i n 30

m i n u t e s a f t e r a u x i n t r e a t m e n t . The s i m i l a r i t y i n k i n e t i c s o f t h e

i n c r e a s e s i n a c t i v i t y o f t h e s y n t h a s e , and o f t h e e a r l y r e s p o n d i n g

mRNAs s u g g e s t s t h a t one o f t h e s e e a r l y r e s p o n d i n g mRNAs may code f o r

13-1, 4 - g l u c a n s y n t h a s e , c a u s i n g t h i s enzyme t o i n c r e a s e by de novo

s y n t h e s i s i n r e s p o n s e t o a u x i n .

C a lc iu m e x h i b i t s a d u a l e f f e c t upon g r o w t h . B u r s t r o m (1957)

r e p o r t e d t h a t c a l c i u m d e f i c i e n t t i s s u e n o t o n l y e x h i b i t s a r e d u c e d

g rowth r a t e bu t a l s o a p p e a r e d t o be more r i g i d . High ,

n o n - p h y s i o l o g i c a l c o n c e n t r a t i o n s o f c a l c i u m (20 mM) a r e known t o

i n h i b i t g rowth r a p i d l y b o t h i n t h e a b s e n c e and p r e s e n c e o f a u x i n .

+2Tagawa and Bonner (1957) s u g g e s t e d t h a t Ca may f u n c t i o n as a

s t i f f e n i n g a g e n t by fo rm in g b r i d g e s b e tw e en c a r b o x y l g r o u p s p r e s e n t

w i t h i n t h e c e l l w a l l . However , C l e l a n d and R a y l e (1977) d e m o n s t r a t e d

t h a t a d d i t i o n o f c a l c i u m t o an mi v i t r o c e l l w a l l p r e p a r a t i o n o f o a t

c o l e o p t i l e t i s s u e k i l l e d by f r e e z i n g and t h a w in g does n o t a l t e r t h e

c e l l w a l l e x t e n s i b i l i t y . I t would a p p e a r t h a t c a l c i u m does n o t

r i g i d i f y t h e c e l l w a l l by f o rm in g c r o s s - l i n k s b e tw e en c e l l w a l l

components i n a n o n - b i o c h e m i c a l p r o c e s s . I n t h e p r e s e n c e o f a u x i n ,

c a l c i u m r e d u c e s b o t h t h e g r o w th r a t e and t h e r a t e o f H+ s e c r e t i o n

(Cohen and N a d l e r , 1976 ) . However , b o t h p r o c e s s e s a r e s t i l l

s t i m u l a t e d n e a r l y t h r e e f o l d above t h e l e v e l s o f g ro w th and

a c i d i f i c a t i o n m ea su red in t h e p r e s e n c e o f c a l c i u m a l o n e ( C l e l a n d and

R a y l e , 1977 ) . The a b i l i t y o f a c i d i c b u f f e r s t o i n d u c e e x t e n s i o n i n

f r o z e n / t h a w e d c o l e o p t i l e t i s s u e was a l s o m o d i f i e d by c a l c i u m .

P r e - t r e a t m e n t o f F /T t i s s u e w i t h 3 mM c a l c i u m i n h i b i t e d a c i d - i n d u c e d

e x t e n s i o n . T h i s c a l c i u m i n h i b i t i o n was r e v e r s i b l e , s i n c e rem ova l o f

t h e c a l c i u m r e s u l t e d i n r a p i d a c i d - i n d u c e d e x t e n s i o n o f t h e c e l l w a l l

m a t e r i a l . Based on t h i s e v i d e n c e , C l e l a n d and R a y le s u g g e s t e d t h a t

c a l c i u m m o d i f i e s a b i o c h e m i c a l component o f c e l l w a l l l o o s e n i n g and

does n o t f u n c t i o n by d i r e c t c h e m i c a l m o d i f i c a t i o n o f t h e c e l l w a l l

components ( c r o s s - b r i d g e s ) . I n c o n t r a s t t o n o n - p h y s i o l o g i c a l l y h i g h

c o n c e n t r a t i o n s (20 mM), lower c o n c e n t r a t i o n s ( 0 . 3 - 1 mM) o f c a l c i u m

s t i m u l a t e a u x i n - i n d u c e d a c i d i f i c a t i o n .

C a lc iu m a p p a r e n t l y a c t s t o m o d i fy b o t h a u x i n - and a c i d - i n d u c e d

g rowth by r e g u l a t i o n o f t h e b i o c h e m i c a l mechanism o f g r o w t h . Due t o

t h e r e l a t i v e l a c k o f i n f o r m a t i o n c o n c e r n i n g t h e i n v o lv e m e n t o f c a l c i u m

i n t h e m o d i f i c a t i o n o f r o o t g r o w t h , t h e c u r r e n t work has been

u n d e r t a k e n t o c h a r a c t e r i z e t h e e f f e c t o f c a l c i u m upon endogenous and

a c i d - i n d u c e d g row th i n m aize r o o t s .

M a t e r i a l s and M e th o d s .

Measurement o f G row th .

Root g ro w th was e s t i m a t e d by d e t e r m i n i n g t h e o v e r a l l i n c r e a s e in

l e n g t h o f t h e r o o t . Growth o f t h e r o o t i s l i m i t e d p r i m a r i l y t o a

s m a l l r e g i o n o f t h e r o o t i d e n t i f i e d as t h e e l o n g a t i o n z o n e . T h i s

r e g i o n u s u a l l y does n o t e x c e e d 1 cm and i s l o c a t e d w i t h i n 1 .5 cm o f

58

t h e r o o t t i p . T h u s , m easu rem en t o f o v e r a l l e x t e n s i o n c l o s e l y

a p p r o x i m a t e s t h e i n c r e a s e i n l e n g t h o f t h o s e c e l l s and t i s s u e s l o c a t e d

w i t h i n t h e e l o n g a t i o n z o n e . Growth was m easu red u s i n g an a n g u l a r

p o s i t i o n t r a n s d u c e r as p r e v i o u s l y d e s c r i b e d ( E va ns , 1976; Kuzmanoff ,

1979 ) . As t h e r o o t e x t e n d s ( g r o w s ) , t h e arm o f t h e t r a n s d u c e r i s

d i s p l a c e d , c h a n g in g t h e v o l t a g e o u t p u t o f t h e t r a n s d u c e r . The change

i n v o l t a g e was m o n i t o r e d by a l i n e a r s t r i p c h a r t r e c o r d e r ( c a l i b r a t i o n

1 mm f u l l s c a l e d i s p l a c e m e n t , + / - 0 . 0 1 mm r e p e a t a b i l i t y ) and by an

I m s a i PCS-42 m ic r o c o m p u te r e q u ip p e d w i t h an 8 b i t a n a l o g / d i g i t a l

c o n v e r t e r (Cromemco D+7A I / O ) . The t r a n s d u c e r s i g n a l was d i g i t i z e d by

t h e A/D c o n v e r t e r , and t h e a v e r a g e o f 500 m easu rem en ts was s t o r e d on

d i s k f o r l a t e r a n a l y s i s e v e r y 20 s e c o n d s . Measurements were a n a l y z e d

i n r e a l t im e f o r s t a n d a r d d e v i a t i o n and s t a n d a r d e r r o r . Measurements

e x c e e d i n g 5% s t a n d a r d e r r o r were r e j e c t e d and r e m e a s u r e d . A l th o u g h

a v e r a g e d v a l u e s were s t o r e d e v e r y 20 s e c o n d s , t h e a c t u a l t im e p e r i o d

o f measurem ent was a b o u t 3 s e c o n d s . A d d i t i o n a l d a t a m a n i p u l a t i o n s ,

which i n c l u d e d c a l c u l a t i o n o f " i n s t a n t a n e o u s r a t e " ( c h a n g e i n r a t e

be tw e en s u c c e s s i v e m e a s u r e m e n t s ) and r e a l t im e g r a p h i n g o f t h e l e n g t h

v s . t im e c u r v e were p e r f o r m e d by t h e c om pu te r d u r i n g t h e r e m a i n i n g 17

s e c o n d s . C a l c u l a t i o n o f g r o w th r a t e was p e r f o r m e d a ) m a n u a l ly by

m easurem ent o f t h e s l o p e o f t h e s t r i p c h a r t r e c o r d e r t r a c i n g and b) by

t h e c om pute r f rom t h e r e c o r d e d v a l u e s . A d d i t i o n a l a n a l y s i s o f t h e

m easu rem en ts were p e r f o r m e d on a TRS-80 model I m ic r o c o m p u te r .

Measurements o f r a t e and l e n g t h were a l s o o b t a i n e d from e x i s t i n g

59

s t r i p c h a r t r e c o r d e r t r a c i n g s u s i n g an x , y d i g i t i z e r w i t h a r e s o l u t i o n

o f 100 i n c r e m e n t s / i n c h .

P r e p a r a t i o n o f S o l u t i o n s .

I n o r d e r t o t e s t t h e d e p e n d en c e o f r o o t g row th upon c a l c i u m ,

r o o t s were t r e a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f c a l c i u m , w i t h t h e

c a l c i u m c h e l a t o r s EDTA and EGTA, and w i t h i n h i b i t o r s o f t h e c a l c i u m

r e g u l a t e d p r o t e i n m o d u l a t o r , c a l m o d u l i n ( e . g . c h l o r p r o m a z i n e ,

t r i f l u o p e r a z i n e ) . The s o l u t i o n b a t h i n g t h e r o o t s was e i t h e r m o d i f i e d

H o a g l a n d ' s s o l u t i o n (Meyer e t a l . , 1955) or m o d i f i e d H o a g l a n d ' s

s o l u t i o n w i t h 5 mM MOPS. The m o d i f i e d H o a g l a n d ' s s o l u t i o n c o n s i s t e d

o f Ca(N03 ) 2 ( 1 . 5 mM); MgS04> KH2P04> KN03 (1 mM e a c h ) ; H3B03 (20 pM) ;

MnCl2 ( 3 . 8 pM); ZnCl2 ( 0 . 3 6 pM); Mo03 ( 0 . 1 8 pM); CuCl2 ( 0 . 1 4 pM). A l l

b a t h i n g s o l u t i o n s were a d j u s t e d t o pH 6 . 5 by t h e a d d i t i o n o f HCl or

NaOH. C a l c iu m was s u p p l i e d as c a l c i u m c h l o r i d e . The t e s t compounds

( c a l c i u m , EGTA, EDTA, c h l o r p r o m a z i n e , t r i f l u o p e r a z i n e ) were added t o

t h e b a t h i n g s o l u t i o n e i t h e r as a c o n c e n t r a t e o r t h e b a t h i n g medium was

p r e p a r e d c o n t a i n i n g t h e t e s t compound and exchanged f o r t h e i n i t i a l

( p r e - t r e a t m e n t ) b a t h i n g medium.

60

P la n t M a te r ia l

Maize s e e d l i n g s ( Zea mays L . h y b r i d WF9 X MS38; C u s to m a iz e ,

Momence, 1 1 1 . , USA) u s e d were g e r m i n a t e d as d e s c r i b e d e a r l i e r ( s e e

C h a p t . 1, M a t e r i a l s and M e th o d s ) .


T r e a t m e n t o f m a iz e r o o t s w i t h c a l c i u m i n c o n c e n t r a t i o n s from

5 - 50 mM r e s u l t e d i n an i n i t i a l d e c r e a s e i n g r o w th r a t e ( F i g . 9 ) .

The d e c r e a s e was r a p i d and began w i t h i n 60 s e c o n d s , a s d e t e r m i n e d by

c om pu te r a n a l y s i s o f l e n g t h v s . t im e d a t a . The i n h i b i t i o n o f g row th

in d u ce d by c a l c i u m was t r a n s i e n t , w i t h t h e d e g r e e o f r e c o v e r y b e i n g

c o n c e n t r a t i o n d e p e n d e n t ( T a b l e 2 ) . At c o n c e n t r a t i o n s from 10 - 50 mM,

r o o t s o f m a ize be g a n t o r e c o v e r f rom t h e i n h i b i t i o n w i t h i n 20 m i n u t e s .

The g ro w th r a t e f o l l o w i n g r e c o v e r y f rom i n h i b i t i o n by c a l c i u m

c o n c e n t r a t i o n s l e s s t h a n 20 mM was g r e a t e r t h a n t h e i n i t i a l r a t e . The

e x t e n t t o which t h e g ro w th r a t e was e nha nce d above t h e i n i t i a l r a t e in

r o o t s r e c o v e r i n g f rom t r a n s i e n t i n h i b i t i o n by low c o n c e n t r a t i o n s o f

c a l c i u m ( l e s s t h a n 20 mM) v a r i e d w i t h t h e c o n c e n t r a t i o n o f c a l c i u m

a p p l i e d ( T a b le 3 ) . The t im e c o u r s e o f r e c o v e r y from i n h i b i t i o n by

c a l c i u m was a l s o c o n c e n t r a t i o n d e p e n d e n t . Com par i son o f t h e s l o p e s

c a l c u l a t e d f o r t im e p e r i o d s o f 10 m i n u t e s a f t e r a d d i t i o n o f c a l c i u m

shows t h a t a d r a m a t i c change from a s t i m u l a t o r y t o an i n h i b i t o r y

e f f e c t o f c a l c i u m o c c u r r e d b e tw e en 5 and 10 mM.

F ig u r e 9.

Com par i son o f t h e E f f e c t o f I n c r e a s i n g C o n c e n t r a t i o n s o f C a lc iu m Upon t h e Growth o f Maize S e e d l i n g R o o t s .

The f i g u r e i s a c o m p o s i t e o f s e v e r a l c u r v e s o b t a i n e d from d i g i t i z e d m e a s u re m e n ts (20 s e c o n d i n t e r v a l s ) o f l e n g t h u s i n g t h e I m s a i m i c r o c o m p u t e r . Growth i s p r e s e n t e d as an i n c r e a s e i n l e n g t h (mm) o f t h e e n t i r e r o o t w i t h t i m e . Each c u r v e i s t h e c o m p o s i t e o f s e v e r a l e x p e r i m e n t s ( s e e T a b l e 2 ) . The m ea su rem e n ts were merged and a v e r a g e d u s i n g a 5 p o i n t l i n e a r moving a v e r a g e ( E r i c k s o n , 19 7 6 ) . Some o f t h e p rog ra m s u t i l i z e d a r e p r e s e n t e d i n Append ixes H - K. N o n - l i n e a r c u r v e f i t t i n g ( G e r a l d , 1978) t e c h n i q u e s ( p o l y n o m i a l and c u b i c s p l i n e ) were t h e n u s e d t o c a l c u l a t e t h e c u r v e s p r e s e n t e d . Each c u r v e c o n t a i n s a t l e a s t 9 ,0 0 0 p o i n t s . The s t a n d a r d e r r o r f o r a l l c u r v e s i s l e s s t h a n 0 . 7 %. The t im e a t which c a l c i u m t r e a t m e n t s were s t a r t e d ha s been a d j u s t e d t o z e r o t im e and z e r o l e n g t h f o r e a s e o f v i s u a l i z a t i o n .

61

Len

gth

(mm

)

10aoasso

- 20 0 20 40 60Minutes

O 'N3

F ig u r e 9.

T ab le 2.

Com par i son o f t h e E f f e c t o f C a lc ium C o n c e n t r a t i o n Upon t h e I n h i b i t i o n and R e c o v e ry o f t h e Growth R a t e o f Maize R o o t s .

Each c a l c i u m c o n c e n t r a t i o n i s r e p r e s e n t e d by 5 e x p e r i m e n t s (3 f o r 15 mM) w i t h a s t a n d a r d e r r o r o f no more t h a n + / - 0 . 5 % c a l c u l a t e d from t h e mean s l o p e v a l u e s p r e s e n t e d i n T a b l e 3 . The l a g t i m e s r e p r e s e n t t h e t im e r e q u i r e d t o r e a c h 50 o r 90 % i n h i b i t i o n a f t e r a d d i t i o n o f c a l c i u m . The t im e r e q u i r e d f o r 50 % i n h i b i t i o n o f t h e p r e - t r e a t m e n t g row th r a t e i s a p p r o x i m a t e l y 1 . 5 m i n u t e s . Only c o n c e n t r a t i o n s g r e a t e r t h a n 20 mM i n h i b i t e d g ro w th by a t l e a s t 90 %. The s e e m i n g l y p a r a d o x i c a l r e c o v e r y from maximal i n h b i t i o n a t h i g h c o n c e n t r a t i o n s o f c a l c i u m s u g g e s t s t h a t t h i s r e c o v e r y i s n o t an a d a p t a t i o n t o c a l c i u m , b u t r a t h e r an a d a p t a t i o n t o h i g h e x t e r n a l o s m o t i c p o t e n t i a l as p r e v i o u s l y r e p o r t e d (Kuzmanoff and E v a n s , 19 8 1 ) . The t im e f o r r e c o v e r y l i s t e d i n t h e t a b l e i s t h e t im e r e q u i r e d a f t e r a d d i t i o n o f c a l c i u m , f o r t h e e s t a b l i s h m e n t o f a s t e a d y g r o w th r a t e .The maximum % o f t h e c o n t r o l f o r t h e r e c o v e r y i s t h e s t e a d y g rowth r a t e a f t e r r e c o v e r y a s a p e r c e n t a g e o f t h e p r e - t r e a t m e n t g r o w th r a t e . The p r e - t r e a t m e n t r a t e was s t a n d a r d i z e d as 100 %.

63

T able 2.

Acid E f f e c t C a lc ium I n h i b i t i o n(mM) Lag t i m e , min .

50% 90%

(+) 5 -------- -----

(+) 10 1 - 2 -----

(+) 15 1 - 2 -----

(+) 20 1 - 2 -----

(+) 25 1 - 2 -----

(+) 50 1 - 2 2

(+) 100 1 - 2 1

MaximumI n h i b i t i o n

R ecove ry t im e , min . (Max % c o n t r o l )

12 % 26 (160 %)

60 % 41 (130 %)

75 % — ------

80 % 38 (80 %)

80 % 38 (80 %)

75-90 % 45 (75 %)

100 % 65 (63 %)

O'-P~

T able 3 .

Com par i son o f C a l c u l a t e d Growth R a t e B e f o r e and A f t e r A d d i t i o n o f C a l c i u m .

Growth r a t e s were a p p r o x i m a t e d by t h e s l o p e o f t h e b e s t f i t l i n e a r r e g r e s s i o n c u r v e c a l c u l a t e d from m u l t i p l e e x p e r i m e n t s . The g r o w th c u r v e s were o b t a i n e d from d a t a o f l e n g t h v s . t im e r e c o r d e d by t h e I m s a i m i c r o c o m p u t e r . The l i n e a r r e g r e s s i o n c a l c u l a t i o n ( u s i n g t h e p rog ram LINREG/BAS) was made a f t e r t h e d a t a had b e e n merged i n t o a s i n g l e d a t a s e t . The d a t a f o r t h e c u r v e s p r e s e n t e d i n F i g u r e 9 were u s e d f o r t h e l i n e a r r e g r e s s i o n c a l c u l a t i o n . The e f f e c t o f c a l c i u m upon g rowth r a t e i s shown as t h e r a t i o o f t h e s l o p e s a f t e r and b e f o r e a d d i t i o n o f c a l c i u m . The s l o p e o f t h e c u r v e i s d e n o t e d by m. The c o r r e l a t i o n c o e f f i c i e n t ( r ) i s a m e a s u r e o f g o odne ss o f f i t ( + / - 1 = 100%). The v a l u e s f o r t h e s l o p e and c o r r e l a t i o n c o e f f i c i e n t ( u p p e r p o r t i o n o f t h e t a b l e ) were c a l c u l a t e d f o r a 90 min . t im e p e r i o d a f t e r t h e a d d i t i o n o f c a l c i u m . The low e r p a r t o f t h e t a b l e p r e s e n t s a c o m p a r i s o n be tw e en s l o p e s c a l c u l a t e d f o r 90 and 10 min t im e p e r i o d s a f t e r a d d i t i o n . The m ag n i tu d e o f s h o r t and lo n g t e r m i n h i b i t i o n by c a l c i u m i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s . The number o f c u r v e s ( e x p e r i m e n t s ) i s g i v e n by n and i n c l u d e s a l l e x p e r i m e n t s , i r r e s p e c t i v e o f t h e d e g r e e e o f d e v i a t i o n r e p r e s e n t e d from t h e mean. Ea c h e x p e r i m e n t c o n t a i n e d 600 - 1200 m e a s u r e m e n t s .

65

66

T a b l e 3.

C a lc iu m m ( b e f o r e ) r m ( a f t e r ) r n

5 mM 0.0 3 3 3 0 .9 9 5 2 0 .0 3 0 2 0 .9943 71

10 mM 0.0 3 2 2 0 .9 9 7 8 0 .0 3 6 2 0 .9 933 44

20 mM 0.0 5 1 8 0 .9 9 4 5 0 .0 2 9 2 0 .9945 33

25 mM 0.0 4 6 3 0 .9 9 9 7 0 .0 291 0 .9901 16

50 mM 0 .0 4 0 4 0 .9 9 8 6 0 .0 1 3 9 0 .9542 7

C a lc ium .. • ar a t i o ( a f t e r / b e f o r e ) br a t i o ( a f t e r / b e f o r e )

5 mM 0 .9 0 7 1.243

10 mM 1.1 2 4 0 .621

20 mM 0 .5 6 4 0 .241

25 mM 0.6 2 9 0 .2 7 4

50 mM 0 .3 4 4 0 .146

a Time p e r i o d was 90 min .

b Time p e r i o d was 10 min.

67

C o n c e n t r a t i o n s o f c a l c i u m g r e a t e r t h a n 10 mM r e s u l t e d i n i n c r e a s i n g

i n h i b i t i o n o f g row th i m m e d i a t e l y a f t e r a d d i t i o n o f c a l c i u m ( w i t h i n

10 m i n u t e s ) . M easuremen ts o v e r a 90 m in u t e p e r i o d i n d i c a t e d an

a p p a r e n t l y anomolous s t i m u l a t i o n o f g row th by 10 mM c a l c i u m . T h e r e i s

a h i g h d e g r e e o f c o r r e l a t i o n o f t h e c a l c u l a t e d s t r a i g h t l i n e w i t h

m ea su rem e n ts o v e r a 90 m i n u t e t im e c o u r s e ( g r e a t e r t h a n 0 . 9 9 ) f o r a l l

c o n c e n t r a t i o n s o f c a l c i u m u s e d . The s t r o n g s h o r t - t e r m i n h i b i t i o n o f

g row th f o r c o n c e n t r a t i o n s g r e a t e r t h a n 5 mM ( T a b le 3 ) , i m p l i e s t h a t

t h e r e a s o n t h e s t r a i g h t l i n e a p p r o x i m a t i o n f i t s i s t h a t t h e g rowth

r a t e o f t h e t i s s u e s h i f t e d r a p i d l y a b o u t a mean v a l u e and t h a t t h e

m ag n i tu d e o f t h e ch a n g es i n r a t e d e c r e a s e d .

M easurements b a s e d upon t h e no rm a l l e n g t h v s . t im e method o f

r e c o r d i n g g row th do n o t r e v e a l t h e c o m p l e x i t y o f t h e p a t t e r n o f change

i n t h e g row th r a t e f o l l o w i n g a d d i t i o n o f c a l c i u m t o t h e b a t h i n g

medium. E x a m i n a t i o n o f t h e c a l c u l a t e d g ro w th r a t e i n d i c a t e s t h a t

a d d i t i o n o f low c o n c e n t r a t i o n s o f c a l c i u m (5 mM) r e s u l t e d i n a c y c l i c

i n h i b i t i o n / r e c o v e r y p a t t e r n i n t h e g row th r a t e ( F i g . 1 0 ) . T h i s

p a t t e r n r e s e m b l e d t h e p a t t e r n o f a damped o s c i l l a t o r . The d e g r e e , i n

b o t h m a g n i tu d e and d u r a t i o n , o f e a c h c y c l e o f t h e o s c i l l a t i o n s ( b o t h

i n h i b i t i o n and t h e s u b s e q u e n t r e c o v e r y ) a p p e a r e d t o d e c r e a s e , u n t i l a

r e l a t i v e l y s t a b l e r a t e o f g ro w th was r e a c h e d . The r o o t t i s s u e

a p p a r e n t l y " a t t e m p t s t o c o m p e n s a t e " f o r t h e i n c r e a s e d e x t e r n a l c a l c i u m

c o n c e n t r a t i o n , and i n t h e p r o c e s s o f a d j u s t m e n t ( a d a p t a t i o n ) , an o v e r

c o m p e n s a t i o n o c c u r s .

F ig u r e 10.

R a p id O s c i l l a t i o n s i n t h e C a l c i u m - I n d u c e d I n h i b i t i o n o f Root Growth .

The e f f e c t o f 5 mM c a l c i u m upon g ro w th r a t e i s p r e s e n t e d i n a c o m p o s i t e g r a p h . Rap id f l u c t u a t i o n s i n t h e g ro w th r a t e o c c u r r e d a f t e r t h e a d d i t i o n o f 5 mM c a l c i u m . A f t e r maximal i n h i b i t i o n , t h e g row th r a t e o s c i l l a t e d a b o u t a m i d p o i n t o f i n c r e a s i n g v a l u e . The f l u c t u a t i o n s d i m i n i s h e d as t h e f i n a l s t e a d y r a t e was a p p r o a c h e d . The p r e s e n t e d c u r v e s were d e r i v e d from t h e c o m b i n a t i o n o f 7 r e p r e s e n t a t i v e e x p e r i m e n t s and c o n t a i n 1305 c om pu te r b a s e d m e a s u r e m e n ts . The g row th r a t e c u r v e was c a l c u l a t e d from t h e l e n g t h v s . t im e c u r v e and smoothed w i t h a 5 p o i n t l i n e a r moving a v e r a g e p r i o r t o c u r v e f i t t i n g . S t a n d a r d e r r o r d i d n o t ex c ee d 2 .1 %.

68

Figure 10

Growth Rate (mm/hr) — — Length (mm)8 Pcno oo

Iroo

o

o>o

ooo

69

70

At h i g h e r c o n c e n t r a t i o n s o f c a l c i u m ( g r e a t e r t h a n 10 mM), a

s i m i l a r p a t t e r n o f i n h i b i t i o n and r e c o v e r y was o b s e r v e d ( F i g . 11 ) .

The d u r a t i o n o f e a c h i n h i b i t i o n / r e c o v e r y c y c l e i n c r e a s e d w i t h

c o n c e n t r a t i o n and t h e m a g n i tu d e o f t h e f i n a l r e c o v e r y d e c r e a s e d .

C a lc iu m c o n c e n t r a t i o n s g r e a t e r t h a n 15 mM r e s u l t e d i n an o v e r a l l

i n h i b i t i o n o f g r o w t h . The f i n a l s t e a d y r a t e o f e l o n g a t i o n was l e s s

t h a n t h a t p r i o r t o a d d i t i o n o f c a l c i u m ( T a b le 3 ) .

I n h i b i t i o n o f g row th by c a l c i u m d i d n o t a p p e a r t o be an o sm o t ic

e f f e c t . The maximum o s m o l a l i t y o f a 10 mM C a C ^ s o l u t i o n would be no

g r e a t e r t h a n 30 m i l l i o s m o l a r . T r e a t m e n t w i t h 50 mM m a n n i t o l d i d n o t

r e s u l t i n any s i g n i f i c a n t i n h i b i t i o n o f g row th ( F i g . 1 2 , 1 3 ) . Osmotic

c o n c e n t r a t i o n s o f 100 - 300 mM m a n n i t o l have b e e n shown t o i n d u ce

r a p i d s h r i n k a g e o f r o o t t i s s u e (Kuzmanoff , 1979; 1 9 8 1 ) . T h u s , t h e

e f f e c t o f C a C ^ c o n c e n t r a t i o n s e x c e e d i n g 35 mM ( c a . 100 mOsM) would be

e x p e c t e d t o c o n t a i n an o s m o t i c component . However , t r e a t m e n t w i t h low

c o n c e n t r a t i o n s o f t h e osmot icum, m a n n i t o l (100 - 300 mM), i n d u ce d an

i n c r e a s e i n g row th r a t e a f t e r t h e i n i t i a l o m s o t i c s h r i n k a g e . Such an

i n c r e a s e i n t h e g ro w th r a t e d i d n o t o c c u r w i t h e q u i v a l e n t o s m o t i c

c o n c e n t r a t i o n s o f c a l c i u m c h l o r i d e ( c f . F i g s . 1 1 , 1 3 ) . T h i s i n d i c a t e s

t h a t t h e p r i m a r y e f f e c t o f c a l c i u m i s n o t o s m o t i c i n n a t u r e . The

sudden i n c r e a s e i n e x t e r n a l f r e e c a l c i u m may p e r t u r b t h e i n t e r n a l f r e e

c a l c i u m c o n c e n t r a t i o n . A l l c a l c i u m c o n c e n t r a t i o n s examined in d u c e d a

r a p i d i n i t i a l i n h i b i t i o n o f r o o t g r o w t h . S i n c e i n h i b i t i o n by c a l c i u m

o c c u r r e d w i t h i n t h e f i r s t 20 - 60 s e c o n d s , i t p r o b a b l y r e s u l t s f rom an

F ig u r e 11.

Com par i son o f t h e E f f e c t o f C a lc iu m Upon t h e C a l c u l a t e d ' I n s t a n t a n t e o u s 1 Growth R a t e o f Maize R o o t s .

The r a t e s , d e r i v e d from t h e m ea s u rem e n ts u s e d i n F i g u r e 9, were c a l c u l a t e d f rom 20 s e c o n d i n t e r v a l s and t h e n smoothed u s i n g a d i g i t a l B u t t e r w o r t h f i l t e r ( m o d i f i e d from Dynacomp I n c . , R o c h e s t e r , N . Y . ) .T h i s f i l t e r i n g t e c h n i q u e removes a l a r g e p o r t i o n o f t h e o s c i l l a t i o n s o f t h e t y p e o b s e r v e d i n F i g u r e 10. A l th o u g h t h e u s e o f t h e d i g i t a l f i l t e r i s v a l i d , and makes t h e d a t a p l o t t a b l e , i t s u s e may mask r e a l c h a n g es ( c f . F i g s . 1 0 , 1 1 ) . Cubic s p l i n e c u r v e f i t t i n g was u s e d t o g e n e r a t e t h e c u r v e s p r e s e n t e d . The o s c i l l a t o r y p a t t e r n p r e s e n t a t low c o n c e n t r a t i o n s o f c a l c i u m d i m i n i s h e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f c a l c i u m . The p a t t e r n was a l s o o b s e r v e d t o d i s s i p a t e w i t h i n c r e a s i n g t im e as t h e r o o t a p p r o a c h e d a new e q u i l i b r i u m w i t h t h e e x t e r n a l f r e e c a l c i u m .

71

Figure 11

G r o w t h Rate (mm/hr)p00 O) INJ ro

te

i

25 ’cS’v>

o

03O

OO

2Z

F ig u r e 12.

E f f e c t o f 50 mM M a n n i t o l Upon Root Growth .

A s l i g h t i n h i b i t i o n o f g ro w th o c c u r r e d in r e s p o n s e t o a p p l i c a t i o n o f t h e o s m o t i c a g e n t m a n n i t o l ( f i r s t a r r o w ) . I n h i b i t i o n o f g row th was s m a l l and t h e r o o t a p p e a r e d t o a d a p t t o t h e i n c r e a s e d e x t e r n a l o s m o t i c p o t e n t i a l . Removal o f m a n n i t o l ( s e c o n d a r r o w ) r e s u l t e d i n a s l i g h t i n c r e a s e i n g row th r a t e p r i o r t o a r e t u r n t o t h e p r e - t r e a t m e n t r a t e . The c u r v e p r e s e n t e d was d e r i v e d f rom t h e c o m p i le d m easu rm en ts o f 6 e x p e r i m e n t s . The s t a n d a r d e r r o r d i d n o t ex c ee d 1 .1 4 %. The e x p e r i m e n t s were merged and smoothed f o r c a l c u l a t i o n o f g r o w th r a t e u s i n g a 5 p o i n t l i n e a r moving a v e r a g e ( E r i c k s o n , 1976 ) .

73

Leng

th

(mm

)

0.9

0.6 1/2 Meyer's

50 mM Mannitol

0.3

0 35 70 140105 175Minutes

F igu re 12.-P*

F ig u r e 13.

C om par i son o f I n h i b i t i o n o f Growth by M a n n i t o l and C a l c i u m .

C a lc iu m a t 10 mM, which h a s an o s m o l a r i t y o f a b o u t 30 mOsm, i n h i b i t e d m a iz e r o o t g rowth more s e v e r e l y t h a n a 50 mM m a n n i t o l s o l u t i o n . The m a n n i t o l g ro w th r a t e c u r v e was c a l c u l a t e d from t h e c om pu te r m ea su rem e n ts o f l e n g t h v s . t im e u s e d i n F i g u r e 12. The g ro w th r a t e c u r v e f o r 5 mM c a l c i u m was d e r i v e d from t h e m easu rem en ts u s e d in F i g u r e 9, and was c a l c u l a t e d f rom 20 s e c o n d i n t e r v a l s and t h e n smoo thed u s i n g a B u t t e r w o r t h d i g i t a l f i l t e r .

75

Gro

wth

Ra

te (m

m/h

r)2.7

50 mM Mannitol

1/2 Meyer's1.5

0.9

0.3

200 40 60 80 100Minutes

F igu re 13.

77

e f f e c t o f t h e e x t e r n a l c a l c i u m e i t h e r w i t h i n t h e c e l l w a l l o r a t t h e

p lasm a membrane.

C o n c e n t r a t i o n s o f c a l c i u m as low as 0 . 5 mM were a l s o examined .

The g e n e r a l p a t t e r n f o r t h e e f f e c t o f low c o n c e n t r a t i o n s

( 0 . 5 - 2 . 5 mM) o f c a l c i u m on g r o w th a l s o a p p e a r e d t o be r a p i d

i n h i b i t i o n and r e c o v e r y . At t h e s e c o n c e n t r a t i o n s e x t r e m e l y r a p i d

o s c i l l a t i o n s i n g r o w th r a t e were o b s e r v e d and may have r e s u l t e d from

some unknown a r t i f a c t s . A l t h o u g h t h e g e n e r a l p a t t e r n o f i n h i b i t i o n

and r e c o v e r y a p p e a r e d t o o c c u r a t c a l c i u m c o n c e n t r a t i o n s as low as

0 . 5 mM, no r e p r o d u c i b l e e f f e c t was o b s e r v e d f o r c a l c i u m c o n c e n t r a t i o n s

be low 2 . 5 mM. T r e a t m e n t w i t h c a l c i u m a t 5 mM r e s u l t e d i n an i n i t i a l ,

r a p i d i n h i b i t i o n f o l l o w e d by r e c o v e r y w i t h i n 4 - 1 0 min. Dur ing t h e

f i r s t 10 - 15 min o f r e c o v e r y , r o o t s t r e a t e d w i t h 5 mM C a C ^ e x h i b i t e d

a r a p i d f l u c t u a t i o n i n g row th r a t e . The g row th r a t e c o n t i n u e d t o

i n c r e a s e a f t e r t h e i n i t i a l i n h i b i t i o n , b u t t h e r a t e changed and

a p p e a r e d t o o v e r s h o o t some i n c r e a s i n g mean v a l u e . As t h e g row th r a t e

c o n t i n u e d t o i n c r e a s e , e v e n t u a l l y above t h e p r e - t r e a t m e n t r a t e , t h e

m a g n i tu d e o f t h e f l u c t u a t i o n s ( o s c i l l a t i o n s ) d i m i n i s h e d .

The o s c i l l a t o r y p a t t e r n may, i n p a r t , be an a r t i f a c t r e s u l t i n g

e i t h e r f rom e l e c t r i c a l n o i s e d u r i n g t h e com pute r a c q u i s i t i o n o f v a l u e s

o r , more l i k e l y , a r e s u l t o f t h e l i m i t e d r e s o l u t i o n o f t h e m e a s u r in g

s y s t e m . A l th o u g h t h e c om pu te r a c q u i s i t i o n o f d i s c r e t e m easu rem en ts

u s e d i n t h i s s t u d y i s more r a p i d t h a n p r e v i o u s l y r e p o r t e d m e th o d s , i t

i s l i m i t e d b y t h e r e l a t i v e l y l o n g t im e p e r i o d s and by t h e number o f

78

i n c r e m e n t s . Based upon t h e t im e i n t e r v a l o f m ea s u rem e n t , and r a n g e

u se d f o r measurem ent o f r o o t g r o w th (1 mm f u l l s c a l e , 20 s e c

i n t e r v a l ) , t h e c o m p u te r m easu rem en t i s l i m i t e d t o a maximum r e s o l u t i o n

o f a b o u t + / - 0 .0 0 5 mm ( f o r a b o u t 200 i n c r e m e n t s u s i n g t h e 8 b i t A/D

c o n v e r t e r i n t h e c o m p u t e r ) . S i n c e g ro w th r a t e s a r e c a l c u l a t e d from

t h e c hange i n l e n g t h w i t h t im e and most commonly e x p r e s s e d w i t h u n i t s

o f mm/hr, t h e l a r g e s t i n h e r e n t e r r o r can be + / - 0 . 9 mm/hr i f a

0 .0 0 5 mm change i n l e n g t h i s m ea s u red w i t h i n a 20 se c t im e p e r i o d .

Such a l a r g e e r r o r i s i n h e r e n t l y i m p r o b a b le b e c a u s e o f t h e a v e r a g i n g

methods u s e d f o r d a t a a c q u i s i t i o n i n t h i s s y s t e m . T h u s , an e r r o r i n

t h e c a l c u l a t e d g row th r a t e i s compounded by any e r r o r i n t h e

measu rem en t o f l e n g t h . E r r o r s i n suc h m ea s u rem e n ts were m in im iz e d by

u s i n g m u l t i p l e m ea su rem e n ts t o c a l c u l a t e b o t h a mean v a l u e and a

p e r c e n t s t a n d a r d e r r o r . The r a p i d f l u c t u a t i o n i n g ro w th r a t e ,

a l t h o u g h n o t e n t i r e l y r e p r o d u c i b l e , most l i k e l y r e p r e s e n t s a r e a l

component o f t h e r e s p o n s e o f m a iz e r o o t s t o low c o n c e n t r a t i o n s o f

c a l c i u m . T h i s c o n c l u s i o n i s b a s e d on two o b s e r v a t i o n s . The f i r s t

o b s e r v a t i o n i s t h a t t h e method o f m easu rem en t a l l o w s an i n c r e a s e d

maximum s e n s i t i v i t y . The p r o b a b i l i t y o f l a r g e f l u c t u a t i o n s

( + / - 0 . 9 mm/hr) i s s m a l l ( 0 . 2 %). The s e c o n d o b s e r v a t i o n i s t h a t

s i m i l a r , a l t h o u g h s m a l l e r and l e s s r a p i d , f l u c t u a t i o n s i n g ro w th r a t e

were a p p a r e n t a t h i g h e r c a l c i u m c o n c e n t r a t i o n s . The p r e s e n c e o f

r e p r o d u c i b l e o s c i l l a t i o n s i n d i c a t e s t h a t t h e r a p i d v a r i a t i o n s o b s e r v e d

a t low c o n c e n t r a t i o n d i d n o t r e s u l t e n t i r e l y f rom a r t i f a c t . The

79

d i f f i c u l t y i n o b t a i n i n g r e p r o d u c i b l e o s c i l l a t o r y p a t t e r n s d u r i n g

t r e a t m e n t w i t h low c o n c e n t r a t i o n o f c a l c i u m i n d i c a t e s t h a t t h i s

com pu te r b a s e d m e a s u r i n g s y s t e m i s o p e r a t i n g a t t h e l i m i t s o f i t s

r e s o l u t i o n . A l a r g e r number o f m ea s u rem e n ts i n a s h o r t e r t im e

i n t e r v a l , and a h i g h e r s e n s i t i v i t y ( a l a r g e r number o f inc rem en ts /m m )

a p p e a r n e c e s s a r y t o r e s o l v e t h e q u e s t i o n o f how low c o n c e n t r a t i o n s o f

c a l c i u m a f f e c t t h e i n i t i a l g rowth r a t e .

No E f f e c t o f C a lc iu m Upon A c i d - S t i m u l a t e d G ro w th .

No i n h i b i t i o n o f a c i d - i n d u c e d m a iz e r o o t g ro w th was found u s i n g

c a l c i u m c o n c e n t r a t i o n s o f 20 mM. Even 100 mM c a l c i u m d i d n o t i n h i b i t

a c i d - s t i m u l a t e d m a iz e r o o t g rowth ( F i g . 1 4 ) .

E f f e c t o f C a l m o d u l in I n h i b i t o r s Upon G row th .

Many s y s t e m s which a r e r e g u l a t e d by, o r d e p e n d e n t upon c a l c i u m

have b e e n shown t o be m e d i a t e d by c a l m o d u l i n (Cheung, 1 9 8 0 ) . S i n c e

r o o t g r o w th i s s t r o n g l y a f f e c t e d by e x t e r n a l c a l c i u m , and t h e c a l c i u m

c h e l a t o r s EDTA and EGTA can mimic a c i d - i n d u c e d e x t e n s i o n , t h e e f f e c t

o f t h e c a l m o d u l i n i n h i b i t o r s c h l o r p r o m a z i n e (CPZ) and t r i f l u o p e r a z i n e

(TFP) on r o o t g ro w th was exam ined .

F ig u r e 14.

A c id S t i m u l a t i o n o f Root Growth i n t h e P r e s e n c e o f C a l c i u m .

R e p r e s e n t a t i v e c u r v e s show t h e l a c k o f i n h i b i t i o n by c a l c i u m o f a c i d - i n d u c e d g row th i n m aize r o o t s . P r e - t r e a t m e n t o f i n t a c t m aize r o o t s w i t h 20 mM o r 100 mM ( f i r s t a r r o w ) c a l c i u m i n h i b i t e d g r o w t h . At 20 mM c a l c i u m t h e i n h i b i t i o n was n o t as s e v e r e as t h a t o b s e r v e d f o r 100 mM c a l c i u m . A c i d i f i c a t i o n o f t h e s o l u t i o n b a t h i n g t h e r o o t t o pH 4 . 0 r e s u l t e d i n a r a p i d i n c r e a s e i n g r o w t h r a t e . The a c i d - i n d u c e d g r o w t h r a t e was n o t i n h i b i t e d by e i t h e r 20 o r 100 mM c a l c i u m .

80

Leng

th

(mm

)

0.1 mmpH * 4

100 mM

pH - 4CaCI

20 mM

0 60 8 020 40 100Minutes

F igu re 14.

82

Low c o n c e n t r a t i o n s o f CPZ ( l e s s t h a n 10 pM) a p p a r e n t l y d i d no t

i n h i b i t t h e g rowth r a t e o f r o o t s ( T a b l e 4 ) . At c o n c e n t r a t i o n s o f

50 pM, c h l o r p r o m a z i n e s t r o n g l y i n h i b i t e d r o o t g rowth a f t e r a l a g p e r i o d

o f a p p r o x i m a t e l y 30 m i n u t e s ( F i g . 1 5 ) . I n h i b i t i o n o f g row th c o n t i n u e d

t o i n c r e a s e d u r i n g t h e f o l l o w i n g h o u r , u n t i l t h e g row th r a t e was

r e d u c e d t o l e s s t h a n 10 % o f t h e p r e - t r e a t m e n t r a t e . At

c o n c e n t r a t i o n s o f 50 pM o r h i g h e r , no r e c o v e r y i n t h e p r e s e n c e o f CPZ

was o b s e r v e d . I f t h e s e e d l i n g was r e t u r n e d t o t h e p r e - t r e a t m e n t

s o l u t i o n (5 mM MOPS, pH 6 . 5 ) , t h e g ro w th r a t e i n c r e a s e d a f t e r a l ag

p e r i o d o f 1 . 5 - 2 . 0 h o u r s . The g ro w th r a t e c o n t i n u e d t o i n c r e a s e

d u r i n g t h e f o l l o w i n g 30 m i n u t e s u n t i l t h e p r e - t r e a t m e n t r a t e was

a t t a i n e d .

I n t h e p r e s e n c e o f low c o n c e n t r a t i o n s o f CPZ ( be low 50 pM), t h e

g row th r a t e d i d r e c o v e r . However , f o r c o n c e n t r a t i o n s a t and above

10 pM t h e f i n a l g ro w th r a t e a f t e r r e c o v e r y was lower t h a n t h e r a t e

p r i o r t o a d d i t i o n o f CPZ. C h l o r p r o m a z i n e a t a c o n c e n t r a t i o n o f 50 pM

i n h i b i t s CaM s t i m u l a t i o n o f p h o s p h o d i e s t e r a s e by 90 %

(Klee e t a l . , 1980 ) . C h l o r p r o m a z i n e a t 50 pM a l s o i n h i b i t e d m aize r o o t

g row th by 90 %. T r i f l u o p e r a z i n e ha s b e e n r e p o r t e d t o be a more p o t e n t

c a l m o d u l i n i n h i b i t o r t h a n CPZ (Weiss e t a l . , 1 9 8 0 ) . Even low

c o n c e n t r a t i o n s o f TFP (10 pM) s t r o n g l y i n h i b i t e d r o o t g row th w i t h a

m a g n i tu d e and l a g t im e o f i n h i b i t i o n s i m i l a r t o t h a t o b s e r v e d f o r 50 pM

CPZ ( F i g . 1 5 ) .

T able 4 .

I n h i b i t i o n o f Maize Roo t Growth by C h l o r p r o m a z i n e .

The p h e n o t h i a z i n e d e r i v a t i v e c h l o r p r o m a z i n e (CPZ) i s a p o t e n t , a l t h o u g h n o n - s p e c i f i c (Weiss e t a l . , 19 8 0 ) , i n h i b i t o r o f c a l m o d u l i n . The e f f e c t o f CPZ upon i n t a c t r o o t g ro w th a t d i f f e r e n t c o n c e n t r a t i o n s was exam ined . C o n c e n t r a t i o n s b e lo w 10 pM d i d n o t i n h i b i t r o o t g r o w th . C h l o r p r o m a z i n e a t 10 pM, i n h i b i t e d g row th by a b o u t 55 - 65 %. Roo ts t r e a t e d w i t h t h i s c o n c e n t r a t i o n o f CPZ r e c o v e r e d f rom i n h i b i t i o n . The s t e a d y r a t e a f t e r r e c o v e r y was a b o u t 80 % o f t h e p r e - t r e a t m e n t g row th r a t e . S t r o n g i n h i b i t i o n o f g ro w th r e s u l t e d from t r e a t m e n t w i t h CPZ a t c o n c e n t r a t i o n s g r e a t e r t h a n 10 pM. The l a g t im e i s t 'ne t im e r e q u i r e d t o r e a c h 50 and 90 % i n h i b i t i o n . Very r a p i d , c o m p l e t e i n h i b i t i o n r e s u l t e d f rom 0 . 1 and 1 . 0 mM CPZ. R e c o v e ry r e f e r s t o an i n c r e a s e i n t h e g row th r a t e above t h e r a t e o f maximal i n h i b i t i o n i n t h e p r e s e n c e o f c h l o r p r o m a z i n e . P r e s e n t e d v a l u e s a r e t h e a v e r a g e o f a t l e a s t 3 m e a s u r e m e n t s .

83

84

T a b l e 4 .

C h l o r p r o m a z i n e Lag Time (min) R e c o v e ryc o n c e n t r a t i o n (mM) 50 % 90%

0 . 0 0 1 a "75" ----- (+)

0 . 0 0 5 a "82" ----- (+)

0 .0 1 0 36 170 (+)

0 .0 5 0 40 65 ( - )

0 .1 0 0 7 7 ( - )

1 .000 7 7 ( - )

I n h i b i t i o n was n o t r e p r o d u c i b l e ,

k R e c o v e ry was i n c o m p l e t e ( c a . 80 % o f p r e - t r e a t m e n t r a t e ) .

F ig u r e 15.

I n h i b i t i o n o f Root Growth by C a l m o d u l in I n h i b i t o r s .

Both c h l o r p r o m a z i n e (CPZ) and t r i f l u o p e r a z i n e (TFP) a r e p o t e n t i n h i b i t o r s o f c a l m o d u l i n . A p p l i c a t i o n o f CPZ a t c o n c e n t r a t i o n s o f 50 and 100 pM s t r o n g l y i n h i b i t e d r o o t g r o w t h , w h i l e 5 pM CPZ a p p e a r e d t o h a v e no i n h i b i t o r y e f f e c t . T r i f l u o p e r a z i n e , wh ich h a s b e e n r e p o r t e d t o be a more p o t e n t i n h i b i t o r o f c a l m o d u l i n t h a n c h l o r p r o m a z i n e(Weiss e t a l . 1980) i n h i b i t e d r o o t g r o w th a t a c o n c e n t r a t i o n o f 10 pM.The f i g u r e i s a c o m p o s i t e o f s e v e r a l c u r v e s o b t a i n e d from d i g i t i z e d m ea su rem e n ts o f l e n g t h u s i n g t h e I m s a i m ic r o c o m p u t e r . Each g rowth c u r v e p r e s e n t e d r e p r e s e n t s a t l e a s t t h r e e e x p e r i m e n t s . The m ea s u rem e n ts f rom s e p a r a t e e x p e r i m e n t s were smoothed u s i n g a 5 p o i n t l i n e a r moving a v e r a g e ( E r i c k s o n , 19 7 6 ) , and merged i n t o a s i n g l e d a t a f i l e . The s t a n d a r d e r r o r f o r a l l c u r v e s p r e s e n t e d i s l e s s t h a n 2 . 8 %.The r o o t l e n g t h and t im e a t which CPZ o r TFP were added was a r b i t r a r i l y a d j u s t e d t o z e r o l e n g t h and z e r o t im e b e f o r e t h e m erg ing o f t h e c u r v e s .

85

Leng

th

(mm

)SuM CPZ

2 .0

10||M TFP

1.0

100|lM CPZ

C P Z ,TF P

- 1.0

Figure 15. -so o 50 100 150 200Minutes

87

C h i o r p r o m a z i n e i n h i b i t i o n o f CaM i s c a l c i u m d e p e n d e n t

(K lee e t a l . , 1 9 8 0 ) , e x h i b i t i n g s t r o n g e r i n h i b i t i o n o f CaM i n t h e

p r e s e n c e o f c a l c i u m . S i n c e low c o n c e n t r a t i o n s o f CPZ a p p a r e n t l y d i d

n o t i n h i b i t r o o t g r o w th , t h e i n h i b i t o r y e f f e c t o f 5 and 10 pM CPZ was

r e - e x a m i n e d a f t e r p r e - t r e a t m e n t o f t h e r o o t s w i t h c a l c i u m .

A p p l i c a t i o n o f 5 pM CPZ a f t e r p r e - t r e a t m e n t w i t h c o n c e n t r a t i o n s o f

c a l c i u m w i t h l i t t l e o r no long t e r m i n h i b i t o r y e f f e c t on g row th

r e s u l t e d i n an i n i t i a l s m a l l i n h i b i t i o n o f g ro w th which o c c u r r e d

w i t h i n 5 - 1 0 m i n u t e s a f t e r a d d i t i o n o f CPZ. T h i s i n h i b i t i o n was

f o l l o w e d by a t r a n s i e n t s t i m u l a t i o n and t h e n by r a p i d , p r o l o n g e d

i n h i b i t i o n o f g ro w th ( F i g . 1 6 ) . The f i n a l g ro w th r a t e was s t a b l e a t

a b o u t 50 % o f t h e p r e - t r e a t m e n t r a t e (40 % o f t h e s t e a d y r a t e a f t e r

c a l c i u m a p p l i c a t i o n ) . A s i m i l a r i n h i b i t i o n w i t h o u t any o b s e r v a b l e

s t i m u l a t i o n , o c c u r r e d when 10 pM CPZ was a p p l i e d . A summary o f t h e

e f f e c t s o f c h l o r p r o m a z i n e upon r o o t g ro w th i s p r e s e n t e d i n T a b l e 4 .

I n h i b i t i o n o f g row th by CaM i n h i b i t o r s m ig h t be a t t r i b u t e d t o

i n h i b i t i o n o f r e s p i r a t i o n , s i n c e g row th ha s been c l e a r l y shown t o be

d e p e n d e n t upon r e s p i r a t i o n ( C l e l a n d , 1967b ) . P r e l i m i n a r y e x p e r i m e n t s

w i t h c o n c e n t r a t i o n s o f CPZ and TFP which s t r o n g l y i n h i b i t g row th

(50 and 25 pM, r e s p e c t i v e l y ) showed t h a t t h e s e l e v e l s o f CPZ and TFP

d i d n o t i n h i b i t r e s p i r a t i o n ( d a t a n o t shown) . I n f a c t , t h e r e a p p e a r e d

t o be a s l i g h t s t i m u l a t i o n o f t h e r e s p i r a t i o n r a t e ( a b o u t 10 %) a f t e r

a 10 - 15 m in u t e l a g . A d d i t i o n o f CPZ o r TFP t o m i t o c h o n d r i a l

F ig u r e 16.

I n h i b i t i o n o f Root Growth by C h l o r p r o m a z i n e i n t h e P r e s e n c e o f C a l c i u m .

C h lo r p ro m a z i n e (CPZ) a t c o n c e n t r a t i o n s o f 5 and 10 pM d i d n o t s i g n i f i c a n t l y i n h i b i t m a iz e r o o t g ro w th ( s e e T a b l e 4 ) . However , b o t h c o n c e n t r a t i o n s o f CPZ s t r o n g l y i n h i b i t e d r o o t g ro w th i f a p p l i e d ( s e c o n d a r r o w ) a f t e r p r e - t r e a t m e n t w i t h c a l c i u m ( f i r s t a r r o w ) . The c u r v e s p r e s e n t e d were c a l c u l a t e d from d i g i t i z e d m ea su rem e n ts o f s t r i p c h a r t r e c o r d i n g s u s i n g an x , y d i g i t i z e r . Each c u r v e r e p r e s e n t s t h r e e e x p e r i m e n t s which were merged i n t o one f i l e and smoothed w i t h a B u t t e r w o r t h d i g i t a l f i l t e r .

88

Gro

wth

Ra

te (m

m/h

r)10 uM • 5 uM CPZ ft

320 mM 1 0 mM CaCU

2

1

0

-50 0 50 100 150Minutes

F igu re 16.

oovo

90

p r e p a r a t i o n s from m aize r o o t s a l s o f a i l e d t o i n h i b i t r e s p i r a t i o n ( d a t a

n o t show n) .

I n summary, m easu rem en ts o f g r o w th o f m aize r o o t s t r e a t e d w i t h

c a l c i u m show t h a t c a l c i u m c o n c e n t r a t i o n s b e low 10 mM s t i m u l a t e r o o t

g rowth a f t e r an i n i t i a l i n h i b i t i o n . A l l c o n c e n t r a t i o n s o f c a l c i u m

from 5 - 100 mM i n i t i a l l y i n h i b i t g r o w th and t h e i n h i b i t i o n i s

i n d e p e n d e n t o f t h e o s m o t i c e f f e c t o f t h e c a l c i u m s o l u t i o n s .

C o n c e n t r a t i o n s a t and above 10 mM c a u s e l o n g - t e r m i n h i b i t i o n o f r o o t

g rowth w i t h t h e d e g r e e o f i n h i b i t i o n i n c r e a s i n g w i t h c o n c e n t r a t i o n .

The g r o w t h r a t e c a l c u l a t i o n s from c om pu te r a c q u i s i t i o n o f l e n g t h v s .

t ime m ea s u rem e n ts r e v e a l t h e p r e s e n c e o f o s c i l l a t i o n s i n t h e g r o w th

r a t e a f t e r a d d i t i o n o f c a l c i u m . The ch a n g es i n g row th r a t e a r e

i n i t i a l l y r a p i d and l a r g e and a p p e a r t o d i s s i p a t e as t h e g ro w th r a t e

a d j u s t s t o a f i n a l s t e a d y r a t e . The o s c i l l a t i o n s i n r a t e a p p e a r t o be

t h e r e s u l t o f t h e g row th mechan ism o v e r - c o m p e n s a t i n g f o r h i g h e x t e r n a l

c a l c i u m c o n c e n t r a t i o n s .

The c a l m o d u l i n i n h i b i t o r s c h l o r p r o m a z i n e and t r i f l u o p e r a z i n e

i n h i b i t g r o w t h , w i t h o u t i n h i b i t i n g r e s p i r a t i o n , a t c o n c e n t r a t i o n s

r e p o r t e d t o i n h i b i t CaM d e p e n d e n t s y s te m s i n a n i m a l s . These

i n h i b i t o r s a r e known t o e x h i b i t s t r o n g e r i n h i b i t i o n i n t h e p r e s e n c e o f

m i l l i m o l a r c a l c i u m c o n c e n t r a t i o n s . . N o n - i n h i b i t o r y c o n c e n t r a t i o n s o f

CPZ i n h i b i t r o o t g row th r a p i d l y when t h e r o o t s have b e e n p r e - t r e a t e d

w i t h c a l c i u m .

91

E x a m i n a t i o n o f c e l l w a l l e x t e n s i b i l i t y showed t h a t t h e c h e l a t o r s

EDTA and EGTA can mimic a c i d - i n d u c e d e x t e n s i o n w h i l e a b o l i s h i n g normal

a c i d - i n d u c e d e x t e n s i o n . The a b i l i t y o f EDTA t o mimic a c i d - i n d u c e d

e x t e n s i o n even a f t e r p r o t e i n d e n a t u r i n g t r e a t m e n t o f t h e r o o t t i s s u e

and t h e l o s s o f a c i d - i n d u c i b l e e x t e n s i o n a f t e r suc h t r e a t m e n t s u g g e s t s

t h a t t h e mechanisms o f EDTA- and a c i d - i n d u c e d e x t e n s i o n a r e

q u a l i t a t i v e l y d i f f e r e n t . E x t e n s i o n r e s u l t i n g from a c i d t r e a t m e n t o f

c e l l w a l l m a t e r i a l i s a p p a r e n t l y d e p e n d e n t upon some component which

i s s e n s i t i v e t o i n a c t i v a t i o n by t r e a t m e n t n o r m a l l y u s e d t o d e n a t u r e

p r o t e i n s . The e f f e c t o f EDTA e x h i b i t s no s u c h d e p e n d e n c e . E x t e n s i o n

i n d u c e d by EDTA a p p a r e n t l y r e s u l t s f rom a d i r e c t c h e l a t i n g e f f e c t upon

t h e c e l l w a l l m a t e r i a l o f m a iz e r o o t s . The r a p i d i n h i b i t i o n o f g row th

by c a l c i u m a t c o n c e n t r a t i o n s g r e a t e r t h a n 10 mM and t h e s t i m u l a t i o n o f

g rowth a t low c o n c e n t r a t i o n (5 mM) s u g g e s t t h a t c a l c i u m may m e d i a t e

c e l l w a l l l o o s e n i n g and g r o w t h by r e g u l a t i n g an enzyme complex w i t h i n

t h e c e l l w a l l o r a t t h e s u r f a c e o f t h e p l a s m a membrane.

The e v i d e n c e t h a t c a l c i u m c h e l a t i n g a g e n t s c a n i n d u c e r a p i d c e l l

e x t e n s i o n i n r o o t s i n d i c a t e s t h a t c a l c i u m b a l a n c e i n r o o t c e l l s p l a y s

a key r o l e i n g o v e r n i n g t h e i r r a t e o f e x t e n s i o n . T h i s i s f u r t h e r

s u p p o r t e d by t h e f i n d i n g s t h a t a p p l i e d c a l c i u m i n d u c e s s t r o n g

o s c i l l a t i o n s i n t h e g ro w th r a t e o f r o o t s and r e s u l t s i n a f i n a l s t e a d y

r a t e w h ic h i s s t r o n g l y c o n c e n t r a t i o n - d e p e n d e n t . These i n d i c a t i o n s o f

a m a jo r r e g u l a t o r y r o l e f o r c a l c i u m i n r o o t g row th r a i s e t h e q u e s t i o n

o f t h e r o l e t h a t c a l m o d u l i n m ig h t p l a y i n g o v e r n i n g t h e g row th p r o c e s s

i n r o o t c e l l s . E v i d e n c e p r e s e n t e d i n t h i s c h a p t e r i n d i c a t e s t h a t

92

i n h i b i t o r s o f c a l m o d u l i n c a n i n h i b i t g row th i n r o o t s w i t h o u t

i n h i b i t i n g r e s p i r a t i o n , and t h a t t h e e f f e c t i v e n e s s o f t h e c a l m o d u l i n

i n h i b i t o r s i s i n f l u e n c e d by t h e l e v e l o f c a l c i u m . T h e s e r e s u l t s a r e

c o n s i s t e n t w i t h t h e s u g g e s t i o n t h a t c a l m o d u l i n may be m e d i a t i n g

c a l c i u m a c t i o n on g r o w th . An a t t e m p t was made t o d e t e c t t h e p r e s e n c e

o f c a l m o d u l i n i n r o o t s o f m a iz e i n o r d e r t o f u r t h e r i n v e s t i g a t e t h i s

p r o p o s a l . S i n c e e a r l i e r work has shown t h a t s u r f a c e p r o t e i n s can be

r e l e a s e d from p l a n t c e l l s by o s m o t i c s h o c k and t h a t l o s s o f t h e s e

p r o t e i n s r e n d e r s t h e c e l l s i n s e n s i t i v e t o a u x i n and t o a c i d , a t t e m p t s

were made t o d e t e c t c a l m o d u l i n b o t h i n o s m o t i c s h o c k p r e p a r a t i o n s from

r o o t s and i n whole r o o t e x t r a c t s .

C h a p t e r I I I . E x a m i n a t i o n o f t h e P r o t e i n a c e o u s M a t e r i a l R e l e a s e d From

Maize R oo t s by Osm oti c S h o c k .


The l o s s o f a c i d - i n d u c i b l e e x t e n s i o n i n f r o z e n thawed m aize r o o t

t i s s u e a f t e r t r e a t m e n t o f t h e t i s s u e by d e n a t u r i n g w i t h t h e c h a o t r o p i c

a g e n t u r e a s u g g e s t s t h a t some p r o t e i n o r p r o t e i n complex may be

n e c e s s a r y f o r a c i d - i n d u c e d g r o w t h . The a b i l i t y o f i n t a c t r o o t s t o

r e c o v e r f rom t r e a t m e n t w i t h u r e a s u g g e s t s t h a t such t r e a t m e n t s do n o t

e x t e n s i v e l y d i s r u p t t h e t i s s u e . A l th o u g h an 8 M u r e a s o l u t i o n r e a d i l y

d e n a t u r e s most p r o t e i n , s u c h a s o l u t i o n a l s o p o s s e s s e s a h i g h o s m o t i c

p o t e n t i a l .

The r e l e a s e o f p r o t e i n by o s m o t i c shoc k f rom t h e c e l l w a l l o r

o u t e r s u r f a c e o f t h e p lasm a membrane o f gram n e g a t i v e b a c t e r i a has

b e e n u s e d t o c h a r a c t e r i z e e x t r a - c e l l u l a r l y l o c a l i z e d p r o t e i n s o f IS.

c o l i (Neu and H e ppe l , 1965; H e p p e l , 1 9 6 7 ,1 9 6 9 ) . R u b i n s t e i n (1977) and

Masuda e t a l . ( 1978) have r e p o r t e d t h a t a u x i n - i n d u c e d g row th i s

i n h i b i t e d by o s m o t i c sh o c k . R u b i n s t e i n has p r o p o s e d t h a t some c e l l

w a l l l o c a l i z e d o r p lasm a membrane bound component o f t h e c e l l w a l l

l o o s e n i n g mechanism i s r e l e a s e d from t h e p l a n t c e l l as a r e s u l t o f

94

o s m o t i c shock and t h a t t h i s component i s e s s e n t i a l f o r a u x i n - i n d u c e d

g row th and a c i d i f i c a t i o n . Osmotic shock has been r e p o r t e d t o i n h i b i t

t r a n s p o r t p r o c e s s e s b e l i e v e d t o i n v o l v e t h e p l a s m a membrane.

I n h i b i t i o n o f g l u c o s e and p h o s p h a t e t r a n s p o r t i n c a r r o t r o o t

(Nieman and W i l l i s , 1971) and i n h i b i t i o n o f ATPase a c t i v i t y f rom

b a r l e y r o o t s ( H a l l and B u t t , 1969) have a l l been a s s o c i a t e d w i t h t h e

o s m o t i c shock r e l e a s e o f p r o t e i n . Gr i inwaldt e t a l . ( 1978 ) have

r e p o r t e d t h a t l i t t l e a p p a r e n t p h y s i c a l damage r e s u l t s f rom o s m o t i c

shock t o m aize r o o t t i s s u e . O s m o t i c a l l y s hoc ked t i s s u e e x h i b i t s

r e d u c e d t r a n s p o r t i m m e d i a t e l y a f t e r shock f o l l o w e d by a r e c o v e r y

r e q u i r i n g 2 - 4 h o u r s . U s in g g e l f i l t r a t i o n t e c h n i q u e s , Schkarenkowa

e t a l . (1978) r e p o r t e d t h a t a t l e a s t t h r e e p r o t e i n a c e o u s components

c o u ld be s e p a r a t e d from t h e m a t e r i a l r e l e a s e d by o s m o t i c shock o f

m aize r o o t s .

A c c o r d in g t o t h e a c i d g rowth h y p o t h e s i s , a u x i n - i n d u c e d

a c i d i f i c a t i o n m e d i a t e s c e l l w a l l l o o s e n i n g . Exogenous a c i d i f i c a t i o n

o f t h e t i s s u e c i r c u m v e n t s t h e n o rm a l a u x i n - i n d u c t i o n o f a c i d i f i c a t i o n

and d i r e c t l y t r i g g e r s some b i o c h e m i c a l c e l l w a l l l o o s e n i n g mechanism.

C a lc iu m d r a m a t i c a l l y p e r t u r b s t h e g ro w th p a t t e r n o f i n t a c t m a ize

r o o t s , and e x o g e n o u s l y a p p l i e d c a l c i u m a t low c o n c e n t r a t i o n s (10 mM

and l e s s ) s t i m u l a t e s , w h i l e h i g h e r c o n c e n t r a t i o n s ( g r e a t e r t h a n 20 mM)

i n h i b i t m aize r o o t g r o w th . The p a t t e r n o f g ro w th i n r e s p o n s e t o

e x t e r n a l c a l c i u m s u g g e s t s t h a t a dynamic e q u i l i b r i u m b e tw e en

c y t o p l a s m i c and e x t r a c e l l u l a r c a l c i u m i s p e r t u r b e d and t h a t

a d j u s t m e n t , w i t h c o n c o m i t a n t r e c o v e r y t o t h e p r e - t r e a t m e n t g rowth

r a t e , r e q u i r e s r e - e s t a b l i s h m e n t o f t h i s e q u i l i b r i u m a c r o s s t h e p lasm a

membrane. The s e c o n d i n t r i g u i n g o b s e r v a t i o n i s t h a t d e n a t u r a t i o n o f

f r o z e n / t h a w e d r o o t t i s s u e , e i t h e r by b o i l i n g m e th a n o l o r u r e a

t r e a t m e n t , a b o l i s h e s a c i d - i n d u c e d , s t r e s s - d e p e n d e n t e x t e n s i o n o f t h e

t i s s u e . The no rm a l i n c r e a s e i n e x t e n s i b i l i t y ( c e l l w a l l l o o s e n i n g )

i n d u c e d by low pH i s l o s t a f t e r t r e a t m e n t t o d i s r u p t p r o t e i n

c o n f o r m a t i o n . However, t h e c h e l a t i n g a g e n t s EDTA and EGTA do i n c r e a s e

c e l l w a l l e x t e n s i b i l i t y a f t e r p r o t e i n d e n a t u r a t i o n and a r e c a p a b l e o f

i n d u c i n g a s eco n d i n c r e a s e i n e x t e n s i b i l i t y a f t e r a c i d - s t i m u l a t e d c e l l

w a l l l o o s e n i n g o f n o n - d e n a t u r e d c e l l w a l l m a t e r i a l . E x a m i n a t i o n o f

t h e d e n a t u r i n g u r e a s o l u t i o n and o t h e r o s m o t i c a g e n t s f o r t h e p r e s e n c e

o f o s m o t i c a l l y r e l e a s e d m a t e r i a l f rom m aize r o o t t i s s u e was u n d e r t a k e n

b a s e d upon t h e t h e s e o b s e r v a t i o n s .

M a t e r i a l s and M e th o d s .

P l a n t M a t e r i a l .

G r a in s o f m aize ( Zea mays L . , h y b r i d WF9 x MS38) were g e r m i n a t e d

as p r e v i o u s l y d e s c r i b e d ( s e e M a t e r i a l s and M ethods , C h a p t . 1 ) .

Soybean ( G l y c i n e max, v a r . " E l f " ) s e e d s were p l a n t e d i n v e r m i c u l i t e

( c a . 6 cm i n d e p t h ) i n p l a s t i c t r a y s (1300 ml v o l u m e ) . The s e e d s were

c o v e r e d w i t h 1 cm o f v e r m i c u l i t e , w a t e r e d w i t h 350 ml o f d e m i n e r a l i z e d

w a t e r , and p l a c e d i n t h e g ro w th chamber . C o n d i t i o n s f o r g e r m i n a t i o n

were as p r e v i o u s l y d e s c r i b e d f o r l e n t i l (Kuzmanoff , 19 7 9 ) .

96

Osmot ic T r e a t m e n t o f T i s s u e f o r P r e l i m i n a r y M e a s u r e m e n t s .

The i n i t i a l e x a m i n a t i o n s f o r t h e r e l e a s e o f m a t e r i a l by o s m o t i c

shock were o f t h e o s m o t i c s o l u t i o n s u s e d t o i n d u c e t h e sh o c k . The

r o o t s o f i n t a c t m a iz e o r s o y b e an s e e d l i n g s were s e q u e n t i a l l y immersed

i n s o l u t i o n s o f 8 M u r e a o r 0 . 6 M m a n n i t o l . The o s m o t i c s o l u t i o n s

were p r e p a r e d i n 10 mM MOPS, and t h e s o l u t i o n s were t h e n a d j u s t e d t o

pH 7 . 0 . The u r e a was p u r i f i e d by e l u t i o n , 2 x , f rom an AG501 x 8

mixed bed column (Bio -R ad L a b o r a t o r i e s ) .

E x t r a c t i o n o f Osmoti c Shock M a t e r i a l .

E x a m i n a t i o n o f t h e components r e l e a s e d by o s m o t i c shock r e q u i r e d

t h e c o l l e c t i o n o f l a r g e r q u a n t i t i e s o f t h e m a t e r i a l . A m o d i f i e d

p r o c e d u r e s i m i l a r t o t h a t r e p o r t e d by R u b i n s t e i n (1982b) was employed

f o r t h e t r e a t m e n t o f r o o t t i s s u e and t h e c o l l e c t i o n o f t h e o s m o t i c

shock m a t e r i a l (OSM). B r i e f l y , a p i c a l r o o t s e g m e n t s , 1 cm i n l e n g t h ,

were c u t and c o l l e c t e d i n an i n c u b a t i o n b u f f e r o f 5 mM HEPES, pH 7 . 0

on i c e f o r p e r i o d s o f 60 m in . At t h e end o f 60 m i n u t e s t h e t i s s u e was

removed f rom t h e i n c u b a t i o n medium and immersed i n t h e o s m o t i c shock

s o l u t i o n o f 0 . 6 M m a n n i t o l , 5 mM HEPES, pH 7 . 0 f o r 15 m i n u t e s . The

t i s s u e was t h e n removed f rom t h e o s m o t i c medium by f i l t r a t i o n on an

open f a c e . B u c h n e r f u n n e l . The t i s s u e was washed w i t h 5 ml o f i c e c o l di

r e h y d r a t i o n b u f f e r (5 mM HEPES, pH 7 . 0 ) w i t h m i l d s u c t i o n a p p l i e d from

a w a t e r a s p i r a t o r , and t r a n s f e r r e d t o 50 ml o f r e h y d r a t i o n b u f f e r f o r

30 m i n u t e s . At t h e end o f t h i s t im e p e r i o d , t h e r e h y d r a t i o n medium

was removed by f i l t r a t i o n and b r o u g h t t o 2 9 . 5 mM B-MCE and 0 .0 1 %

sod ium a z i d e . R e h y d r a t i o n medium was c o l l e c t e d i n volumes o f 500 ml

b e f o r e b e g i n n i n g t h e e x t r a c t i o n o f OSM. I n d i v i d u a l r e h y d r a t i o n medium

a l i q u o t s were combined and f r o z e n a t - 8 0 C u n t i l a t o t a l volume o f 1

l i t e r was a t t a i n e d . P r e l i m i n a r y c o n c e n t r a t i o n o f t h e r e h y d r a t i o n

medium u s e d an Amicon u l t r a f i l t r a t i o n s y s t e m w i t h a YM-10 membrane

( m o l e c u l a r w e i g h t c u t o f f : 1 2 , 0 0 0 ) . The r e h y d r a t i o n medium was

f i l t e r e d t h r o u g h a Whatman GF/D f i l t e r p r i o r t o c o n c e n t r a t i o n . A

t o t a l o f 1 kg o f m a iz e r o o t t i s s u e (1 cm s e g m e n t s ) was t r e a t e d f o r t h e

f i n a l p r e p a r a t i o n . The o s m o t i c t r e a t m e n t s o l u t i o n was a l s o c o l l e c t e d

and s t o r e d i n a s i m i l a r m anne r .


I n i t i a l M easurement o f Osm oti c Shock R e l e a s e d M a t e r i a l .

D ur ing t h e c o u r s e o f e x a m in in g t h e p h y s i c a l p r o p e r t i e s o f t h e

c e l l w a l l s o f r o o t t i s s u e , t h e c h a o t r o p i c and o s m o t i c s o l u t i o n s u s e d

t o t r e a t r o o t s , were examined . The u r e a s o l u t i o n s u s e d f o r p r o t e i n

d e n a t u r a t i o n were examined f o r ch a n g es i n u l t r a v i o l e t s p e c t r a a f t e r

t r e a t m e n t o f t h e r o o t t i s s u e . A l a r g e i n c r e a s e i n a b s o r b a n c e , w i t h a

m a jo r b r o a d peak a t a b o u t 265 nm was o b s e r v e d . A number o f c e l l u l a r

c om ponen t s , i n c l u d i n g p r o t e i n c o u l d be r e s p o n s i b l e f o r t h i s change in

t h e UV a b s o r b a n c e . The a p p e a r a n c e and s i z e o f t h i s peak was d e p e n d e n t

b o t h upon t h e t y p e o f s o l u t i o n s u s e d f o r t r e a t i n g t h e t i s s u e and t h e

98

d u r a t i o n o f t h e t r e a t m e n t . Osm oti c s o l u t i o n s c o n t a i n i n g m a n n i t o l

( 0 . 6 M), o r s o l u t i o n s c o n t a i n i n g c h a o t r o p i c a g e n t s suc h as u r e a (8 M),

r e s u l t e d i n t h e r e l e a s e o f c e l l u l a r component s i n t o t h e t r e a t m e n t

s o l u t i o n . T h i s r e l e a s e was d e t e c t a b l e as an i n c r e a s e i n a b s o r b a n c e a t

265 - 272 nm. U re a (8 M) s o l u t i o n s e x h i b i t e d a n o n - l i n e a r i n c r e a s e in

UV a b s o r b a n c e o v e r a t ime p e r i o d o f 60 min ( F i g . 1 7 ) . P r o l o n g i n g t h e

t r e a t m e n t p e r i o d beyond 40 m i n u t e s r e s u l t e d i n o n l y a s m a l l a d d i t i o n a l

i n c r e a s e i n a b s o r b a n c e . A s i m i l a r UV s p e c t r a l cha nge o c c u r r e d when

0 . 6 M m a n n i t o l was u s e d i n p l a c e o f t h e u r e a s o l u t i o n ( F i g . 1 8 ) . The

r e l e a s e o f c e l l u l a r component s c o n t r i b u t i n g t o t h e i n c r e a s e i n

a b s o r b a n c e d i d n o t o c c u r when w a t e r , m o d i f i e d H o a g l a n d ' s ( M e y e r ' s )

s o l u t i o n , o r 5 mM MOPS were u s e d as t h e t r e a t m e n t s o l u t i o n ( F i g . 18 ) .

The p e a k a b s o r b a n c e o f b o t h u r e a and m a n n i t o l s o l u t i o n s u s e d t o

o s m o t i c a l l y shoc k r o o t t i s s u e d i f f e r e d from t h e UV a b s o r b a n c e o f t h e

p r o t e i n b o v i n e se rum a lbum in (BSA), f r a c t i o n V. Both s o l u t i o n s

e x h i b i t e d s t r o n g , a l b e i t b r o a d p e a k s c e n t e r e d n e a r 280 nm and and a

second peak n e a r 220 nm ( F i g . 1 9 ) . The b r o a d p e a k a t 280 nm was

p r e s e n t i n BSA and i s p r e s e n t i n mos t p r o t e i n s . T h i s a b s o r p t i o n peak

r e s u l t s f rom UV a b s o r p t i o n by t h e a r o m a t i c r i n g o f t h e amino a c i d s

t y r o s i n e , t r y p t o p h a n and p h e n y l a l a n i n e . P e p t i d e bonds a b s o r b a t t h e

lower w a v e l e n g t h s be tw e en 220 - 230 nm. The UV s p e c t r u m o f t h e

m a n n i t o l o s m o t i c s h o c k s o l u t i o n d i f f e r e d f rom b o t h t h e u r e a o s m o t i c

shock s o l u t i o n and t h e BSA sample i n t h a t t h e s e c o n d peak was c e n t e r e d

n e a r 220 and n o t 230 nm. Osmot ic s h o c k o f e i t h e r m a ize o r soybean

F ig u r e 17.

Change i n t h e UV S p ec tru m o f 8 M U rea Used f o r O sm otic Shock a s a F u n c t io n o f Time o f Im m ers ion o f R oot S a m p le s .

I n t a c t r o o t s o f m a ize w ere o s m o t i c a l l y sh o ck ed by im m ers ion in 8 M u r e a (10 mM MOPS, pH 6 . 5 ) . Each r o o t was t r e a t e d s e q u e n t i a l l y f o r t h e t im e i n d i c a t e d . The s p e c t r u m o f t h e u r e a was r e c o r d e d a t t h e end o f t h e t r e a t m e n t . The r e f e r e n c e b l a n k was 8 M u r e a . A l a r g e i n c r e a s e i n t h e a b s o rb a n c e o c c u r r e d w i t h i n 10 m in u t e s , w i t h n e a r l y an e q u a l i n c r e a s e d u r in g t h e seco n d 10 m in u te p e r i o d (20 min m e a su re m e n t) . S m a l l e r i n c r e a s e s i n t h e a b s o rb a n c e o c c u r r e d f o r t im e s g r e a t e r th a n 20 m in u t e s .

99

Abs

orba

nce

(OD)

100

1060

0 9

400.8

0.7

300.6

0.5 20

0.4

0.31 0 mi n

0.2

0.1

0

200 3 2 0240 280 3 6 0Wavelength (nm)

F igu re 17.

F igu re 18.

C om parison o f OSM R e le a s e d by U rea and M a n n i to l T r e a t m e n t .

The s p e c t r a l change o f t h e s o l u t i o n s o f u r e a and m a n n i to l (20 r o o t s / 1 5 ml s o l u t i o n ) was m easu red a f t e r t r e a t i n g t h e r o o t s f o r 20 m in u t e s . Each r o o t was t r e a t e d f o r 20 min i n t u r n . D i f f e r e n c e s in b o th peak p o s i t i o n and m a g n i tu d e o f t h e p e a k s w ere a p p a r e n t . I n c o n t r a s t t o t h e o s m o t ic s o l u t i o n s , t r e a t m e n t w i t h w a te r o r 5 mM MOPS f o r 2 h o u rs d id n o t r e s u l t i n any s i g n i f i c a n t change in s o l u t i o n a b s o rb a n c e p r o f i l e . The c u r v e s f o r b o th w a te r and MOPS t r e a t m e n t s a r e i n d i s t i n g u i s h i b l e .

101

Abs

orba

nce

(OD)

102

0.8

0.7

0.6

0.5 Urea 8 M

0.4

Mannitol 0.6 M0.3

0.2

0.1

Water0

MOPS 5 mM

200 240 280 320 360Wavelength (nm)

F ig u r e 18.

F ig u r e 19.

C om parison o f t h e UV A b s r o p t i o n S p e c t r a o f BSA and OSM from U rea and M a n n i to l T r e a tm e n t S o l u t i o n s .

The b ro a d a b s o r p t i o n peak c e n t e r e d a t 270 - 280 nm was p r e s e n t in a l l t h r e e s o l u t i o n s . The BSA p e a k a t 220 nm was p r e s e n t i n b o th o s m o t ic s o l u t i o n s a f t e r t r e a t m e n t o f m a iz e r o o t t i s s u e (20 r o o t s / 1 5 ml s o l u t i o n ) , how ever t h e a b s o r p t i o n peak was s h i f t e d t o a s l i g h t l y low er w a v e le n g th (210 - 220 nm) i n t h e m a n n i to l s o l u t i o n .

103

Abs

orba

nce

(OD)

08

0.7

0.6

Urea 8M0.5

0.4

Mannitol 0.6M0.3

0.2

BSA 025 mg/ml

3202 00 240 280Wavelength (nm)

F ig u r e 19.

r o o t s r e s u l t e d i n changes i n t h e UV s p e c t r a o f t h e s o l u t i o n u s e d to

a d m i n i s t e r t h e o s m o t ic s h o c k . A peak a p p e a re d b e tw e en 260 and 280 nm.

However, t h e peak a b s o rb a n c e f o r so y b ean was s h i f t e d as com pared to

m a iz e . I n p a r t i c u l a r , t h e a b s o rb a n c e a t low er w a v e le n g th s

(220 - 240 nm) was a b s e n t ( F i g . 2 0 ) . P e p t i d e bonds a b s o rb s t r o n g l y

w i t h i n t h i s r e g i o n and t h e a b s e n c e o f t h i s peak i n t h e o s m o t ic sh o ck

medium f o r soybean r o o t s s u g g e s t s t h a t i n so y b e an t h e m a t e r i a l

r e l e a s e d may n o t c o n t a i n a s i g n i f i c a n t amount o f p r o t e i n . No a s s a y

was p e r fo rm e d t o d e te r m in e p r o t e i n c o n t e n t o f t h e so y b ean o sm o t ic

shock s u b s t a n c e . T h a t s e v e r a l (more th a n one ) com ponents were

r e l e a s e d in r e s p o n s e t o t r e a t m e n t w i t h c h a o t r o p i c o r h ig h o s m o t ic

s o l u t i o n s i s i n d i c a t e d by t h e b ro a d n a t u r e o f t h e p e a k .

P r e l i m i n a r y F r a c t i o n a t i o n o f t h e O sm otic Shock M a t e r i a l .

M a t e r i a l r e l e a s e d from m aize r o o t s by o s m o t ic sh o c k w i t h 8 M u r e a

was f r a c t i o n a t e d by m o le c u la r w e ig h t s i z e e x c l u s i o n c h ro m a to g ra p h y on

a S ephadex G-100 column ( 2 . 5 x 50 cm) e q u i l i b r a t e d w i t h 10 mM T r i s ,

pH 7 . 0 . A t l e a s t t h r e e m o le c u la r w e ig h t c l a s s e s o f com ponents were

p r e s e n t i n t h e o s m o t ic sh o c k m a t e r i a l ( F ig . 2 1 ) . The l a r g e s t peak

a l s o had th e s m a l l e s t m o le c u la r w e ig h t r a n g e and u n d o u b te d ly c o n t a i n e d

th e u r e a from t h e o s m o t ic sh o c k medium. The s i z e o f t h e peak was a

r e s u l t o f t h e u r e a , w hich a b s o r b s s t r o n g l y a t 280 nm. The com ponents

r e p r e s e n t e d by t h e t h r e e p e a k s o b t a i n e d from t h e G-100 e l u t i o n had

F ig u r e 20.

S p e c t r a l Changes o f M a n n i to l S o l u t i o n A f t e r T r e a tm e n t o f M aize and Soybean R o o t s .

T r e a tm e n t o f b o th m aize and so y b e an r o o t s f o r 20 m in u te s ( s e q u e n t i a l t r e a t m e n t ) r e s u l t e d in an i n c r e a s e i n th e UV s p e c t r u m o f t h e o sm o t ic t r e a t m e n t s o l u t i o n s . The s p e c t r u m o f o s m o t ic s o l u t i o n s u s e d f o r t r e a t m e n t o f soybean d i f f e r e d c o n s i d e r a b l y from th e a b s o r p t i o n p r o f i l e o f m aize OSM. The m a jo r , b ro a d peak o f m aize OSM (270 - 280 nm) was s h i f t e d t o a low er w a v e le n g th (265 - 270 nm) in so y b e an OSM. The second peak a t 220 nm i n m aize OSM was m i s s in g in so y b e a n . The soybean OSM peak a t 270 nm was a l s o s h a r p e r t h a n th e 270 - 280 nm peak p r e s e n t i n t h e OSM o f m a iz e .

106

Abs

orba

nce

(OD)

0.7

0.6

Maize0.5

0.4

02 Soybean

0.1

200 240 280 320 360Wavelength (nm)

F igu re 20.

F ig u r e 21.

E l u t i o n P r o f i l e o f M aize OSM from G-100 S ep h ad ex .

The o s m o t ic sh o ck m a t e r i a l o b t a i n e d by t r e a t m e n t o f 100 m aize r o o t s i n 25 ml o f 8 M u r e a , 10 mM MOPS, pH 6 .5 was lo a d e d o n to a G-100 Sephadex column and e l u t e d w i t h 10 mM T r i s , pH 7 . 0 . Two d i s t i n c t p e a k s (# 2 , 3 ) and a p r o b a b le t h i r d peak (# 1) w ere o b t a i n e d .A b so rb a n ce was m o n i to re d a t 280 nm. The column was c a l i b r a t e d u s i n g m o le c u la r w e ig h t s t a n d a r d s a t 0 .5 mg/ml o f BSA, I B - l a c to g l o b u l i n , and c y to c h ro m e C. The l a r g e s t peak (# 3) u n d o u b te d ly i n c l u d e s t h e u r e a f rom th e o s m o t ic s o l u t i o n and may o b s c u r e a d d i t i o n a l p e a k s . The m o le c u la r w e ig h t s t a n d a r d s w ere u s e d t o e s t i m a t e t h e M f o r t h e p e a k s o f th e e l u t i o n p r o f i l e and a r e p r e s e n t e d i n t a b l e 5.

108

Abs

orba

nce

(280

nm)

16

14

12Sephadex G-100

10

\ BSA08

06

04

02

8060 10040200Fractions (ml)

F igu re 21.

a p p ro x im a te m o le c u la r w e ig h ts o f 8 1 .4 , 4 3 . 3 , and 1 1 .5 k i l o d a l t o n s

(T a b le 5 ) . P r o t e i n was p r e s e n t in t h e OSM as m easu red by th e method

o f B r a d fo r d ( 1 9 7 6 ) . O sm otic sh o ck m a t e r i a l was d i a l y z e d o v e r n i g h t

a g a i n s t 10 mM T r i s , pH 7 .0 w i th two c h a n g es o f b u f f e r in o r d e r t o

remove t h e u r e a f rom th e sa m p le . E l u t i o n o f t h e d i a l y z e d OSM from a

Sephadex G-50 column ( 1 .5 x 40 cm) r e v e a l e d t h e p r e s e n c e o f a t l e a s t

f o u r m a jo r peaks w i t h a p r o b a b le f i f t h peak n e a r t h e l e a d i n g edge o f

t h e v o id volume ( F i g . 2 2 ) . E s t i m a t i o n s o f MW i n d i c a t e d a r a t h e r l a r g e

d i s t r i b u t i o n w hich r a n g e d from 1 5 ,1 0 0 - 6 8 ,5 0 0 (T a b le 6 ) . The

o b v io u s o v e r l a p p i n g o f e l u t i o n p e a k s #1 - 4 s u g g e s t e d t h a t a d d i t i o n a l

m inor com ponents w ere masked by t h e l a r g e r p e a k s .

The r e l e a s e , o r l o s s , o f com ponents from t h e c e l l u l a r m a t r i x o f

o s m o t i c a l l y shocked i n t a c t r o o t t i s s u e c o i n c i d e d w i t h t h e a p p a r e n t

l o s s o f s e n s i t i v i t y t o a c i d s t i m u l a t i o n o f g ro w th . E x a m in a t io n o f t h e

o sm o t ic sh o c k m a t e r i a l a f t e r d i a l y s i s and p a r t i a l c o n c e n t r a t i o n

r e v e a l e d t h e p r e s e n c e o f num erous p r o t e i n bands ( P l a t e IV ) . The

a p p e a r a n c e o f s e v e r a l p r o t e i n s in t h e r e h y d r a t i o n medium a t t h e same

t im e t h a t t h e a b i l i t y o f a c i d t o s t i m u l a t e g ro w th was l o s t , i s

c o n s i s t e n t w i th t h e h y p o t h e s i s t h a t a c i d - i n d u c e d g ro w th i s

e n z y m a t i c a l l y m e d ia te d .

A m a jo r r e m a in in g q u e s t i o n was w h e th e r o r n o t t h e OSM c o n ta i n e d a

c a lc iu m b i n d i n g , p e rh a p s c a lc iu m r e g u l a t e d , p r o t e i n .

T ab le 5.

OSM Sephadex G-100 P eaks and T h e i r C a l c u l a t e d M o le c u la r W e ig h t s .

The m id p o in t o f e ach peak o b t a i n e d from th e Sephadex G-100 e l u t i o n o f t h e n o n - d i a l y z e d OSM was m e a s u re d . The m id p o in t s o f t h e t h r e e peaks w ere th e n f i t t e d t o a l i n e a r , l o g a r i t h m i c and se co n d o r d e r e q u a t i o n . M o le c u la r w e ig h t s t a n d a r d s o f b o v in e se rum a lb u m in , f r a c t i o n V (BSA), B - l a c t o g l o b u l i n , and c y to c h ro m e C w ere u s e d t o d e te r m in e t h e m o le c u la r w e ig h t e l u t i o n p r o f i l e o f t h e co lum n. The c a l c u l a t e d e q u a t i o n s a r e :

L i n e a r : y = - 1 .6 1 x + 117

P o ly n o m ia l : y = 0 .0 1 8 1 x ^ - 3 .3 4 2 x + 153 .955

L o g a r i t h m ic : y = - 0 .0 4 8 5 l o g ( x ) + 5 .7 1 1

The p e r c e n t a g e e r r o r f o r t h e c a l c u l a t e d MW o f t h e s t a n d a r d s i s a l s o p r e s e n t e d . The e r r o r was c a l c u l a t e d a s :

% = 100 x a b s o l u t e v a lu e ( ( t r u e MW - c a l c ' d M W )/( tru e MW))

111

T ab le 5 .

Peak # M id p o in t (m l) ̂ C a l c u l a t e d MWx l o g ( x ) x

1 2 2 .1 8 1 .4 1 0 3 .4 8 8 .92 4 5 .8 4 3 .3 3 2 .7 3 8 .83 6 5 .1 1 1 .5 12 .6 13 .1

M o le c u la r W eight Sample

S ta n d a r d MW (Kd) 2

X

% E r r o rl o g ( x ) X

BSA 6 6 . 2 0 .0 0 8 2 .2 7 0 .3 8( J - l a c t o g l o b u l i n 1 8 .0 0 . 1 1 1 1 1 .3 8 .8 3Cytochrom e C 1 2 .4 0 .1 9 9 0 10 .80

F ig u r e 22.

E l u t i o n P r o f i l e o f D ia ly z e d OSM E l u t e d from a G-50 Sephadex Column.

The OSM o b ta in e d by t r e a t m e n t o f 100 m aize r o o t s in 25 ml o f 8 M u r e a , 10 mM MOPS, pH 6 .5 was d i a l y z e d a g a i n s t 10 mM T r i s , pH 7 .0 w i th two changes o f b u f f e r p r i o r t o a p p l i c a t i o n o f t h e OSM sam ple t o t h e G-50 Sephadex colum n. F ou r d i s t i n c t p eak s ( # 2 - 5 ) w ere o b se rv e d w i t h a p r o b a b l e f i f t h peak (# 1 ) p r e s e n t a t t h e l e a d i n g edge o f peak # 2. The colum n was c a l i b r a t e d w i th m o le c u la r w e ig h t s t a n d a r d s o f BSA, 1 3 - la c to g lo b u l in , and c y to ch ro m e C. The c a l c u l a t e d m o le c u la r w e ig h ts o f t h e e l u t e d peaks o f t h e OSM a r e p r e s e n t e d in t a b l e 6 .

113

(UJU

083) e

ou

eq

josq

v

Sephadex G-500.4 BS A

(3-Lac

0.3

Cyt c

Q2

20 1000 40 8060 120

Fractions ( m l )

F igu re 22.

114

T ab le 6 .

OSM S ephadex G-50 P e a k s and T h e i r C a l c u l a t e d M o le c u la r W e ig h t s .

The m id p o in t o f e a c h peak o b t a i n e d from t h e Sephadex G-50 e l u t i o n o f th e d i a l y z e d OSM was m e a s u re d . The m id p o in t s o f t h e f i v e peaks w ere t h e n f i t t e d to a l i n e a r and se co n d o r d e r e q u a t i o n . M o le c u la r w e ig h t s t a n d a r d s o f b o v in e se rum a lb u m in , f r a c t i o n V (BSA), 6- l a c t o g l o b u l i n , and c y to ch ro m e C w ere u s e d t o d e te r m in e t h e m o le c u la r w e ig h t e l u t i o n p r o f i l e o f t h e co lum n. The c a l c u l a t e d e q u a t i o n s a r e :

L i n e a r : y = 0 .7 3 3 x + 7 9 .8 4

P o ly n o m ia l : y - 0 .0 0 5 7 x ^ - 1 .3 6 7 x + 9 0 .6 7 3

The p e r c e n ta g e e r r o r f o r t h e c a l c u l a t e d MW o f t h e s t a n d a r d s i s a l s o p r e s e n t e d . The e r r o r was c a l c u l a t e d a s :

% = 100 x a b s o l u t e v a lu e ( ( t r u e MW - c a l c ' d M W )/( true MW))

115

T ab le 6 .

P eak # M id p o in t (m l) ̂ C a l c u l a t e d MWx l i n e a r

1 1 7 .5 6 8 .5 6 7 .02 3 1 .0 5 3 .8 5 7 .13 4 1 .0 4 4 .2 4 9 .74 4 2 .5 4 2 .9 4 8 .75 8 6 .5 15 .1 1 6 .4

C a l c u l a t e d e r r o r o f t h e f i t t e d c u r v e s .

M o le c u la r W eight S t a n d a r d % E r r o rSample MW (Kd) x l i n e a r

BSA 6 6 .2 0 .0 2 1 .06B - l a c t o g l o b u l i n 1 8 .0 1 .0 0 17 .8 0C ytochrom e C 1 2 .4 2 .1 1 2 0 .6 0

P l a t e IV.

V i s u a l i z a t i o n o f P r o t e i n P r e s e n t i n OSM.

P a r t i a l l y p u r i f i e d a l i q u o t s o f m aize o s m o t ic sh o c k m a t e r i a l c o n t a i n i n g a b o u t 50 n g /1 0 p i o f sam ple were lo a d e d o n to e i t h e r l i n e a r 12 % o r g r a d i e n t p o l y a c r y l a m id e g e l s and v i s u a l i z e d by s i l v e r s t a i n i n g . Lane 1 ,7 : MW s t a n d a r d s . Lane 2: M a t e r i a l e l u t e d from DEAE-Sephadex G-25 w i t h 0 . 4 M NaCl. Lane 3: M a t e r i a l e l u t e d from DEAE-Sephadex G-25 w i t h 0 .6 M NaCl ( a f t e r 0 . 4 M e l u t i o n ) . Lane 4: O sm otic sh o c k m a t e r i a l p r i o r t o l o a d i n g o n to DEAE-Sephadex G-25.L ane 5: M a t e r i a l e l u t e d from DEAE-Sephadex G-25 w i th 0 .8 M NaCl ( a f t e r 0 . 6 M N a C l) . Lane 6 : P u r i f i e d b o v in e c a lm o d u l in . M o le c u la r w e ig h t s t a n d a r d s w ere lysozym e ( 1 4 .4 k d ) , so y b e an t r y p s i n i n h i b i t o r ( 2 1 .5 k d ) , c a r b o n i c a n h y d ra s e ( 3 1 .0 k d ) , ov a lb u m in ( 4 5 .0 k d ) , b o v in e se ru m a lb u m in ( 6 6 .2 k d ) , and p h o s p h o r y l a s e B ( 9 2 .5 k d ) . The p o s i t i o n o f e a ch MW s t a n d a r d i s i n d i c a t e d by a s e r i e s o f c o r r e s p o n d in g b a r s to t h e r i g h t o f l a n e 1 and 7 . A l l sa m p le s w ere p r e p a r e d i n 0 .1 mM C aC ^* The 0 .6 M NaCl e l u t i o n ( l a n e 3) c o n t a i n e d one band ( n e a r 1 4 .4 kd m a rk e r ) w i th a m o b i l i t y s i m i l a r t o t h a t o f a f f i n i t y p u r i f i e d OSM CaM. O n ly p a r t i a l p u r i f i c a t i o n o f t h e OSM was p o s s i b l e u s i n g DEAE co lum ns . F u r t h e r p u r i f i c a t i o n by t h i s p r o c e d u r e was a b a n d o n ed .

117

P l a t e IV.

119

M easurem ent and P u r i f i c a t i o n o f C a lm o d u l in from t h e OSM.

P r e l i m i n a r y m easu rem en ts f o r t h e p r e s e n c e o f c a lm o d u l in (CaM) in

t h e o s m o t ic shock m a t e r i a l w ere p o s i t i v e . C a lm o d u l in c o n t e n t was

m easu red by a p h o s p h o d i e s t e r a s e (PDE) a c t i v a t i o n a s s a y m o d i f ie d from

Sharma and Wang (1979) and d e s c r i b e d e l s e w h e r e ( s e e C h a p te r IV and

A ppendix E ) . The o sm o t ic sh o ck m a t e r i a l was c o n c e n t r a t e d w i th th e

Amicon u l t r a f i l t r a t i o n sy s te m t o 1 /100 (50 m l) o f i t s i n i t i a l vo lum e.

The OSM was th e n d i a l y z e d a g a i n s t 10 mM MOPS, pH 7 .0 , w i th t h r e e

changes o f b u f f e r and a sam ple t o b u f f e r r a t i o o f 1 /1 0 0 . S t i m u l a t i o n

o f PDE above t h e b a s a l ( c o n t r o l ) l e v e l by t h e d i a l y z e d , c o n c e n t r a t e d

OSM was g r e a t e r t h a n 300 %/10 p i o f OSM sam ple ( P l a t e V). However,

n e a r l y one t h i r d o f t h e s t i m u l a t i o n o f PDE by th e o s m o t ic shock

m a t e r i a l was n o t r e v e r s i b l e by s u b s t i t u t i o n o f EGTA f o r c a lc iu m .

S in c e CaM s t i m u l a t i o n o f PDE i s c a lc iu m d e p e n d e n t , a t l e a s t one t h i r d

o f t h e m ea su red a c t i v i t y c o u ld n o t be a t t r i b u t e d t o c a lm o d u l in . The

c a lc iu m in d e p e n d e n t s t i m u l a t i o n o f p h o s p h o d i e s t e r a s e may have been th e

r e s u l t o f p a r t i a l p r o t e o l y s i s o f t h e PDE by t h e OSM. To i n h i b i t any

p r o t e o l y t i c a c t i v i t y p r e s e n t i n t h e o s m o t ic sh o c k m a t e r i a l , t h e

c o n c e n t r a t i o n o f m e r c a p t o e th a n o l was i n c r e a s e d t o 50 mM and th e

c h y m o try p s in i n h i b i t o r PMSF ( p h e n y l m e t h y l s u l f o n y l f l u o r i d e ) was

i n c lu d e d a t 25 p l / 5 0 ml ( @5 0 mg/ml in DMSO) d u r in g a d d i t i o n a l

e x t r a c t i o n s . F u r t h e r p u r i f i c a t i o n was u n d e r t a k e n t o i d e n t i f y and

c o n f i r m t h e p r e s e n c e o f c a lm o d u l in i n t h e o s m o t ic sh o ck medium.

P u r i f i c a t i o n em ployed a p r o c e d u r e s i m i l a r t o t h a t f o r t o t a l CaM

P l a t e V.

A ssay f o r C a lm o d u l in C o n te n t i n M aize OSM.

C a lm o d u l in c o n t e n t was d e te r m in e d by t h e a c t i v a t i o n o f PDE and m e a su re d by t h e r e l e a s e o f i n o r g a n i c p h o s p h a te by t h e F i s k e - S u b a r r o w m ethod as m o d i f i e d from Sharraa and Wang ( 1 9 7 9 ) . D ia ly z e d , c o n c e n t r a t e d o s m o t ic sh o c k m a t e r i a l f rom m aize r o o t s s t i m u l a t e d PDE a c t i v i t y and was c a lc iu m d e p e n d e n t . C u v e t t e 1: C o n t r o l , no OSM. C u v e t t e s 2 - 6 : I n c r e a s i n g amounts o f OSM. The volum es a r e : c u v e t t e 2 : 5 p i , 3 :1 0 p i , 4 :2 0 p i , 5 :4 0 p i , 6 :1 0 0 p i . T o t a l volume o f t h e a s s a y sam ple was m a i n t a i n e d a t 1 m l. C u v e t te 7: C a lc iu m d e p e n d e n t s t i m u l a t i o n o f PDE s t i m u l a t i o n by OSM. C u v e t te 8 : B a s a l PDE a c t i v i t y i n th e p r e s e n c e o f EGTA.

120

P l a t e V.

e x t r a c t i o n ( s e e C h a p te r IV) and u s e d a p h e n o t h i a z i n e a f f i n i t y column

f o r t h e f i n a l i s o l a t i o n o f c a lm o d u l in from t h e OSM ( P l a t e V I ) .

P h o s p h o d i e s t e r a s e a s s a y o f t h i s sam ple e x h i b i t e d s l i g h t l y more th a n

400 % s t i m u l a t i o n o f PDE above th e b a s a l l e v e l and was c o m p le te ly

r e v e r s i b l e by EGTA ( F ig . 2 3 ) . The c a lm o d u l in e x t r a c t e d from t h e OSM

d i f f e r e d from b o v in e CaM i n b o th i t s maximum s t i m u l a t i o n o f PDE and

amount r e q u i r e d t o r e a c h s a t u r a t i o n . U s in g b o v in e b r a i n c a lm o d u l in as

a s t a n d a r d , t h e OSM c a lm o d u l in r e a c h e d s a t u r a t i o n a t a low er l e v e l o f

PDE s t i m u l a t i o n and d id s o more r a p i d l y t h a n b o v in e c a lm o d u l in .

I d e n t i f i c a t i o n o f OSM C a lm o d u l in by R a d io - im m u n o a ssa y .

F u r t h e r i d e n t i f i c a t i o n o f t h e p r e s e n c e o f c a lm o d u l in as one o f

t h e com ponents o f t h e o s m o t ic sh o c k m a t e r i a l was a c c o m p li s h e d by u s e

o f a c a lm o d u l in r a d io - im m u n o a s s a y (New E n g la n d N u c l e a r ) . C a l c u l a t i o n

o f CaM c o n t e n t from m easu rem en ts a t 1 /25 and 1 /5 0 d i l u t i o n o f th e

c a lm o d u l in sam p le p u r i f i e d from t h e OSM showed m aize r o o t CaM t o be

p r e s e n t a t a c o n c e n t r a t i o n o f a b o u t 2 u n i t s / 1 0 p i (T a b le 7 ) . T h is i s

in e x c e l l e n t a g re e m e n t w i th t h e m easu rem en ts o b t a i n e d from t h e

p h o s p h o d i e s t e r a s e a c t i v a t i o n a s s a y . The i s o l a t i o n o f an a p p a r e n t l y

p u re sam ple o f CaM w i th a m o le c u la r w e ig h t s i m i l a r t o t h a t o f b o v in e

c a lm o d u l in and v i r t u a l l y i d e n t i c a l t o c a lm o d u l in e x t r a c t e d from whole

r o o t t i s s u e , and t h e p o s i t i v e a s s e s s m e n t o f CaM c o n t e n t by

r a d io - im m u n o a s s a y c o n f i rm e d t h e p r e s e n c e o f c a lm o d u l in as one o f th e

p r o t e i n s r e l e a s e d from o s m o t i c a l l y shocked t i s s u e .

P l a t e VI.

SDS-PAGE V i s u a l i z a t i o n o f P u r i f i e d OSM C a lm o d u l in .

C a lm o d u l in was i s o l a t e d and p u r f i e d from t h e m aize r o o t o sm o t ic sh o c k m a t e r i a l u s i n g a p r o c e d u r e s i m i l a r t o t h a t em ployed f o r e x t r a c t i o n from w hole r o o t t i s s u e ( s e e C h a p te r IV , M a t e r i a l s and M e th o d s ) . The a f f i n i t y column p u r i f i e d OSM CaM was lo a d e d o n to a 15 % p o l y a c r y la m id e g e l a t a p p r o x im a te ly 30 n g /1 0 p i and v i s u a l i z e d w i th t h e m o d i f i e d s i l v e r s t a i n i n g p r o c e d u r e o f O a k le y ( 1 9 8 0 ) . Lanes 1 ,7 : M o le c u la r w e ig h t s t a n d a r d s as d e s c r i b e d i n P l a t e IV. Lane 2:P u r i f i e d OSM CaM a f t e r p u r i f i c a t i o n two t im e s on th e p h e n o th i a z i n e a f f i n i t y column (a s d e s c r i b e d i n C h a p te r I V ) . Lane 3 ,6 : Bovine CaMs t a n d a r d . Lane 4 : D ia ly z e d , c o n c e n t r a t e d (100 f o l d ) OSM, 20 p i o fs a m p le . Lane 5 : D ia ly z e d , c o n c e n t r a t e d (100 f o l d ) OSM, 10 p i o fsa m p le . L anes 1 ,2 and 3 - 6 a r e from two d i f f e r e n t g e l s . M o le c u la r w e ig h t s t a n d a r d s a re i n d i c a t e d by c o r r e s p o n d in g b a r s t o t h e l e f t o f l a n e 1 and t o t h e r i g h t o f l a n e 7. A p a i r o f bands i n t h e c ru d e OSM o f l a n e s 4 and 5 ( a t a r ro w s ) h a v e m o le c u la r w e ig h ts s i m i l a r t o b o v in e CaM. Based upon whole r o o t e x t r a c t i o n o f CaM, t h e low er band i s p r o b a b l y c a lm o d u l in .

123

P l a t e V I.

F igu re 23.

S t i m u l a t i o n o f PDE by P u r i f i e d OSM C a lm o d u l in .

A f f i n i t y p u r i f i e d OSM c a lm o d u l in from m aize was a s s a y e d f o r s t i m u l a t i o n o f p h o s p h o d i e s t e r a s e a c t i v i t y . Maximum s t i m u l a t i o n o f PDE a c t i v i t y ( s a t u r a t i o n ) by OSM c a lm o d u l in i s n o t a s l a r g e as t h a t o f b o v in e CaM. C o n c e n t r a t i o n o f t h e OSM c a lm o d u l in f o r t u i t o u s l y y i e l d e d a b o u t one u n i t CaM/5 p i .

125

ul of OSM 10 15 20 25 30 35

4 0 0

coOoSB

ma)300

o<Uloa

200

100

t 1--------- 1--------- 1--------- 1--------- 1--------- 1--------- r

CaM

OSM

J I i i i < I I0 1 2 3 4 5 6 7

Units of Calmodulin

F ig u r e 23.

T ab le 7.

Com par i son o f F i t t e d C u rv e s Used t o C a l c u l a t e CaM C o n t e n t o f OSM Us ing R a d io - I m m u n o a s s a y .

L i n e a r , l o g a r i t h m i c , and q u a d r a t i c e q u a t i o n s were f i t t e d t o t h e RIA s t a n d a r d c u r v e . A l th o u g h t h e r e l a t i o n o f c o n c e n t r a t i o n t o p e r c e n t s p e c i f i c b i n d i n g would be e x p e c t e d t o be l o g a r i t h m i c , t h e b e s t f i t o b t a i n e d was q u a d r a t i c . The p e r c e n t a g e e r r o r (% E) o f e a c h f i t t e d c u r v e was c a l c u l a t e d as t h e e r r o r b e tw e e n t h e a c t u a l and c a l c u l a t e d c o n c e n t r a t i o n o f b o v i n e CaM u s e d t o g e n e r a t e t h e c a l i b r a t i o n c u r v e .The c a l c u l a t e d e q u a t i o n s a r e :

L i n e a r : y = - 0 . 2 1 6 x + 2 1 .5

L o g a r i t h m i c y = - 1 7 ( l o g x ) + 78 .412

Q u a d r a t i c y = 0 .0 0 4 2 x - 0 . 8 8 5 x + 4 7 .0 2

127

128

T a b l e 7.

CaM(ng)

%b / bqX

C a l c u l a t e d CaM (ng) %E l o g ( x ) %E 2

X %E

1 0 .0 57 .7 5 9 . 0 9 .8 9 . 4 5 . 8 9 .9 4 0 .5 7

5 .0 72 .35 5 . 9 1 7 .2 5 . 6 11 .8 5 . 0 0 .2 0

2 .5 8 4 .2 0 3 . 3 3 2 . 0 3 . 0 2 0 .4 2 . 3 7 .4 2

1 .25 91 .85 1 .7 3 2 .0 1 .5 2 2 .6 1 .2 3 .6 9

0 .6 3 101 .60 ____ ___ ------- 0 . 5 2 0 .0

129

Measurement o f ATPase a c t i v i t y o f t h e OSM.

Neu and Heppel (1 9 6 5 ) d e m o n s t r a t e d t h a t . o s m o t i c shoc k c o u ld

r e l e a s e p lasm a membrane a s s o c i a t e d ATPases f rom IS. c o l i . One

mechanism by which c a l c i u m , c a l m o d u l i n and H+ c o u l d i n t e r a c t would be

t h r o u g h t h e i r m u tu a l i n t e r a c t i o n w i t h a p l a s m a membrane ATPase. On

t h i s b a s i s , t h e c r u d e OSM was examined f o r ATPase a c t i v i t y . The a s s a y

p r o c e d u r e u s e d (Appendix F) was o b t a i n e d f rom Sigma C hem ica l Co. ( S t .

L o u i s , M o . ) . P r e l i m i n a r y m ea su rem e n ts showed t h e p r e s e n c e o f ATPase

a c t i v i t y i n t h e c r u d e ( f i l t e r e d , c o n c e n t r a t e d ) o s m o t i c s h o c k m a t e r i a l .

The ATPase was o b s e r v e d t o be s t i m u l a t e d by low c o n c e n t r a t i o n s o f

c a l c i u m ( 0 .1 mM) by n e a r l y 70 % and i n h i b i t e d by h i g h e r c a l c i u m

c o n c e n t r a t i o n s (1 mM). ATPase a c t i v i t y o f t h e OSM was a l s o p a r t i a l l y

i n h i b i t e d by EGTA (T a b le 8 ) .

I n summary, o s m o t i c s h o c k o f m a iz e r o o t s r e l e a s e s a l a r g e number

o f p r o t e i n s . R u b i n s t e i n (1982b) h a s shown t h a t t h e p r o t e i n s r e l e a s e d

d u r i n g o s m o t i c s h o c k do n o t o r i g i n a t e f rom t h e c y t o p l a s m . The

p r o t e i n s a p p e a r t o be r e l e a s e d p r i m a r i l y f rom t h e c e l l w a l l and o u t e r

s u r f a c e o f t h e p l a s m a membrane w i t h some p o s s i b l e c o n t r i b u t i o n from

p la s m a d e s m a ta a s s o c i a t e d p r o t e i n s . I n c o n j u n c t i o n w i t h t h e l o s s o f

t h e s e p r o t e i n s , m a iz e r o o t s l o s e t h e i r a b i l i t y t o u n d e rg o

a c i d - s t i m u l a t e d g r o w t h . I n i t i a l e x a m i n a t i o n by g e l f i l t r a t i o n o f

p r o t e i n s r e l e a s e d by o s m o t i c shoc k d e m o n s t r a t e d t h a t a t l e a s t f i v e

d i s c e r n a b l e p r o t e i n p e a k s were p r e s e n t . The peak o f t h e l o w e s t

m o l e c u l a r w e i g h t was a p p r o x i m a t e l y 1 6 ,4 0 0 which c o i n c i d e s w i t h t h e

T able 8 .

P r e l i m i n a r y Measurement o f ATPase A c t i v i t y o f t h e OSM.

The o s m o t i c shock medium was m easu red f o r ATPase a c t i v i t y by a method o b t a i n e d from Sigma Chemica l Co. ( S t . L o u i s , M o . ) . P r e l i m i n a r y m esu rem en ts showed t h e p r e s e n c e o f ATPase a c t i v i t y i n t h e c r u d e OSM. The ATPase was s t i m u l a t e d by low c o n c e n t r a t i o n s o f c a l c i u m ( 0 . 1 mM) a p p r o x i m a t e l y 70 %, and i n h i b i t e d by h i g h (1 mM) c o n c e n t r a t i o n s o f c a l c i u m . ATPase a c t i v i t y was a l s o i n h i b i t e d by EGTA. The ATPase used f o r t h e c o n t r o l was g r a d e IV, o u a b a i n s e n s i t i v e , f rom dog k i d n e y (Sigma # A0142) .

130

131

T a b l e 8 .

Sample + Ca + EGTA % o f C o n t r o l_________________ (mM)_______________(mM)_________________________

ATPase 0 0 100

OSM 0 0 88

OSM 0 .1 0 130

OSM 1 . 0 0 74

OSM 0 0 .1 44

132

r e p o r t e d m o l e c u l a r w e i g h t r a n g e f o r c a l m o d u l i n . Measurements f o r

c a l m o d u l i n c o n t e n t d e m o n s t r a t e d t h e p r e s e n c e o f c a l m o d u l i n - l i k e

a c t i v i t y p r e s e n t i n t h e o s m o t i c s h o c k m a t e r i a l . The p r e s e n c e o f

c a l m o d u l i n as one o f t h e c o n s t i t u e n t s o f t h e o s m o t i c a l l y r e l e a s e d

p r o t e i n s was c o n f i r m e d by r a d i o - i m m u n o a s s a y , p h o s p h o d i e s t e r a s e a s s a y ,

and p u r i f i c a t i o n o f t h e CaM from t h e o s m o t i c shoc k m a t e r i a l . The

c a l m o d u l i n p r e s e n t i n t h e OSM a p p e a r s t o be o f a low er m o l e c u l a r

w e ig h t t h a n b o v i n e c a l m o d u l i n and i d e n t i c a l t o CaM e x t r a c t e d from

whole r o o t t i s s u e .

The p r e s e n c e o f an ATPase as a component’ o f t h e o s m o t i c shock

m a t e r i a l was d e m o n s t r a t e d i n p r e l i m i n a r y m e a s u r e m e n t s . The ATPase

a c t i v i t y a p p e a r s t o be s e n s i t i v e t o c a l c i u m w i t h s t i m u l a t i o n o f

a c t i v i t y by low c o n c e n t r a t i o n s and i n h i b i t i o n o f a c t i v i t y by h i g h

c o n c e n t r a t i o n s o f c a l c i u m . The p r e s e n c e o f c a l m o d u l i n and ATPase

a c t i v i t y i n t h e m a t e r i a l r e l e a s e d by o s m o t i c shock s u p p o r t s t h e

s u g g e s t i o n t h a t t h e mechanism o f a c i d - i n d u c e d g row th i n m aize r o o t s

i n v o l v e s c a l c i u m m e d i a t i o n o f a c a l m o d u l i n r e g u l a t e d p r o t e i n complex .

At l e a s t one o f t h e components r e g u l a t e d by c a l m o d u l i n i s most l i k e l y

a c a l m o d u l i n - d e p e n d e n t ATPase.

C h a p t e r IV. Q u a n t i f i c a t i o n o f E x t r a c t a b l e C a l m o d u l in f rom R oo ts o f

Maize and Le a ves o f S p i n a c h .


The i m p o r t a n c e o f c a l c i u m i n d e v e l o p m e n t a l and p h y s i o l o g i c a l

p r o c e s s e s i n b o t h a n im a l s and p l a n t s ha s b e e n r e c o g n i z e d f o r many

y e a r s ( C u t h b e r t , 1970; S c a r p a and C a r o f o l i , 1 9 7 8 ) . R e c e n t work

i n d i c a t e s t h a t s e v e r a l p r o c e s s e s known t o i n v o l v e c a l c i u m a r e

r e g u l a t e d by c a l c i u m m o d u l a t o r p r o t e i n s (Cheung, 19 8 0 ) . A l l o f t h e s e

+2p r o t e i n s b i n d Ca q u a n t i t a t i v e l y , and in many i n s t a n c e s m o d u la t e t h e

a c t i v i t y o f a s e c o n d p r o t e i n o r p r o t e i n complex . C a lm o d u l in (CaM) i s

a s m a l l ( c a . 1 7 ,0 0 0 d a l t o n s ) , a c i d i c (pK 4 . 3 ) , h i g h l y c o n s e r v e dd

p r o t e i n which i s a member o f t h e g e n e r a l c l a s s o f c a l c i u m m o d u l a t o r

p r o t e i n s which i n c l u d e s S - 1 0 0 , p a r v a l b u m i n , c a l c i n e u r i n and

t r o p o n i n - I . S i n c e t h e i s o l a t i o n and i d e n t i f i c a t i o n o f c a l m o d u l i n by

Cheung (1967 , 1971) and K a k i u c h i e t a l . ( 1 9 7 3 ) , many o f t h e p r o c e s s e s

i n v o l v i n g i o n pumping ( e . g . ATPases) and ho rm ona l r e s p o n s e s

( e . g . e p i n e p h r i n e ) which r e q u i r e c a l c i u m h a v e b e e n shown t o be

d e p e n d e n t upon c a l m o d u l i n . I n p l a n t s , c a l m o d u l i n has b e e n r e p o r t e d t o

be p r e s e n t w i t h i n t h e c h l o r o p l a s t s ( J a r r e t t e t a l . , 1982) where i t may

r e g u l a t e c h l o r o p l a s t NAD k i n a s e (A nderson and C o r m ie r , 1978 ) .

133

134

P h y s i o l o g i c a l s t u d i e s u t i l i z i n g t h e c a l m o d u l i n i n h i b i t o r s

c h l o r p r o m a z i n e and t r i f l u o p e r a z i n e ( s t e l a z i n e ) h a v e i m p l i c a t e d

c a l m o d u l i n i n t h e r e g u l a t i o n o f p l a n t c e l l g r o w t h ( E l l i o t , 1980, 1983;

Kuzmanoff , 1982; E l l i o t e t a l . , 1983) and t h e i n d u c t i o n o f H+

s e c r e t i o n by f u s i c o c c i n (Lado e t a l . , 1981; B o n e t t i e t a l . , 1983) .

I n h i b i t o r s o f c a l m o d u l i n have b e e n shown t o i n h i b i t g r a v i t r o p i s m

(S locum and Roux, 19 8 3 ) , and t h e r e i s i n c r e a s i n g e v i d e n c e t h a t c a l c i u m

r e d i s t r i b u t i o n p l a y s a ke y r o l e i n t h e g r a v i t r o p i c r e s p o n s e

(Lee e t a l . , 19 8 3 ) .

Because of increased interest in the potential role of calmodulin

in the regulation of plant growth, there is an increasing need for a

rapid procedure for extraction and quantification of calmodulin in

plant tissues. In this study, a procedure is described for the

isolation and purification of calmodulin from maize root tissue.

Quantification of the calmodulin content of the tissue was determined

and is also presented.

Materials and Methods.

A l l r e a g e n t s , e x c e p t t h e a f f i n i t y g e l and SDS-PAGE m o l e c u l a r

w e i g h t s t a n d a r d s were o b t a i n e d from Sigma C h em ica l Co. ( S t . L o u i s , Mo.

63178 ) . The F i s k e - S u b a r r o w r e a g e n t (Sigma # 6 6 1 - 8 ) was s u p p l i e d

p r e - m i x e d . The 5 ' n u c l e o t i d a s e (E .C . 3 . 1 . 3 . 5 f rom C r o t a l u s a t r o x

venom, Sigma # N 3880) was p r e p a r e d a t 0 .1 2 5 u n i t s / 1 0 p i i n

10 mM T r i s b a s e , 0 . 5 mM magnesium a c e t a t e , pH 7 . 0 , d i v i d e d i n t o

135

a l i q u o t s o f 1 . 0 ml and s t o r e d a t - 8 0 C.

The a c t i v a t o r d e f i c i e n t p h o s p h o d i e s t e r a s e (PDE), (E .C . 3 . 1 . 4 . 1 7

from b o v i n e h e a r t , Sigma # P0520) u s e d i n t h e a s s a y f o r CaM was k e p t

f r o z e n e i t h e r i n t h e l y o p h y l i z e d s t a t e ( p r i o r t o u s e ) o r p r e p a r e d i n

PDE b u f f e r ( 7 . 5 mM T r i s - H C l , 0 . 3 7 5 mM c a l c i u m c h l o r i d e , 0 .7 5 mM

magnesium a c e t a t e , 0 . 7 5 mM i m i d a z o l e , 25 % w/w g l y c e r o l , pH 7 . 0 ) a t

16 mU/10 p i .

The i n i t i a l b o v i n e c a l m o d u l i n s t a n d a r d was p u r c h a s e d from

B oe h r inge r -M a nnhe im . A d d i t i o n a l b o v i n e s t a n d a r d s were p r e p a r e d by t h e

p r o c e d u r e d e s c r i b e d .

The p h e n o t h i a z i n e a f f i n i t y g e l was p u r c h a s e d from Bio-Rad

L a b o r a t o r i e s (2200 W r ig h t A v e . , Richmond, CA. 94804) .

The m a iz e s e e d l i n g s were u s e d 2 .5 days a f t e r p l a n t i n g . A p i c a l

r o o t s egm en ts 1 . 5 - 2 . 0 cm i n l e n g t h were u s e d f o r t h e e x t r a c t i o n .

The r o o t s egm en ts were p l a c e d i n e x t r a c t i o n b u f f e r (20 mM T r i s - H C l ,

1 mM EDTA, 20 mM fl-MCE, 0 .0 1 % sod ium a z i d e , pH 7 . 5 ) i m m e d i a t e l y a f t e r

c u t t i n g . The r o o t segm en ts were c u t i n s m a l l l o t s w i t h no more t h a n

20 min be tw e en t h e s t a r t o f c u t t i n g and t r a n s f e r t o e x t r a c t i o n b u f f e r .

Fo r c o m p a r a t i v e p u r p o s e s , CaM was a l s o e x t r a c t e d from s p i n a c h

l e a v e s and from b o v i n e b r a i n . The s p i n a c h l e a v e s were d e b l a d e d and

s t o r e d on i c e p r i o r t o e x t r a c t i o n . Bovine b r a i n s were o b t a i n e d from a

l o c a l s l a u g h t e r h o u s e , c l e a n s e d o f b l o o d c l o t s , and s t o r e d on i c e

p r i o r t o e x t r a c t i o n .

136

P la n t M a t e r ia l .

Corn g r a i n s (Zea mays L . h y b r i d WF9 x 38MS; C u s t o m a i z e , Momence,

1 1 1 . , USA) were s oa ked i n d e m i n e r a l i z e d w a t e r f o r 24 h and p l a c e d

b e tw e en wet ( d o u b l e d i s t i l l e d w a t e r ) p a p e r t o w e l s on p l a s t i c t r a y s

mounted i n a v e r t i c a l p o s i t i o n . The s e e d s were g e r m i n a t e d a t room

t e m p e r a t u r e (20 - 24 C) u n d e r f l u o r e s c e n t room l i g h t i n g ( i n t e n s i t y

a p p r o x i m a t e l y 175 + 25 pE/m - s e c ) and u s e d 2 . 5 days a f t e r p l a n t i n g .

F r e s h l y c u t s p i n a c h l e a v e s were o b t a i n e d from a l o c a l m a r k e t .

Assay for CaM Activity and Protein Content.

A m o d i f i e d p h o s p h o d i e s t e r a s e a s s a y s i m i l a r t o t h a t d e s c r i b e d by

Sharma and Wang (1979) was u s e d f o r d e t e r m i n a t i o n o f CaM. The b a s i s

f o r t h e a s s a y i s d e s c r i b e d d i a g r a m a t i c a l l y i n F i g u r e 24. B r i e f l y , t h e

a s s a y s o l u t i o n c o n s i s t e d o f 0 . 1 ml o f a s s a y b u f f e r (360 mM T r i s b a s e ,

360 mM i m i d a z o l e , 45 mM magnes ium a c e t a t e , pH 7 . 5 ) , 20 p i o f 4 . 5 mM

c a l c i u m c h l o r i d e , 20 p i o f 5 ' n u c l e o t i d a s e ( 0 .1 2 5 u n i t s / 1 0 p i ) and 2.5 p i

o f p h o s p h o d i e s t e r a s e (16 mU/10 p i ) . To t h i s was added 10 p i o f t h e

p r o t e i n sample and 0 . 7 ml o f w a t e r . The sample was mixed i n a 1 . 5 ml

Snap-Cap v i a l ( W a l t e r S a r s t e d t I n c . , P r i n c e t o n N . J . , # 7 2 .6 9 0 ) by

v o r t e x i n g b r i e f l y and t h e n i n c u b a t e d f o r 1 min a t 30 C. The a s s a y was

s t a r t e d by a d d in g 50 p i o f 1 0 .8 mM 3 ' : 5 ' cAMP t o t h e sample and

v o r t e x i n g . The sample was t h e n i n c u b a t e d f o r 30 min a t 30 C. The

a s s a y was t e r m i n a t e d by a d d in g 100 p i o f 55 % t r i c h l o r o a c e t i c a c i d and

F ig u r e 24.

Assay Procedure for Calmodulin.

The method u s e d t o d e t e r m i n e t h e p r e s e n c e and amount o f c a l m o d u l i n was d e r i v e d from t h e F i s k e - S u b a r r o w method employed by Sharma and Wang ( 1 9 7 9 ) . The p r o c e d u r e u s e s a c o u p l e d e n z y m a t i c r e a c t i o n and i s b a s e d upon t h e q u a n t i t a t i v e s t i m u l a t i o n o f PDE by c a l m o d u l i n . P h o s p h o d i e s t e r a s e c l e a v e s 3 ' : 5 * cAMP t o p r o d u c e 5* AMP. T h i s p r o d u c t i s c l e a v e d by 5 ' - n u c l e o t i d a s e t o y i e l d a d e n o s i n e and i n o r g a n i c p h o s p h a t e . The p h o s p h a t e i s a s s a y e d by a m o d i f i e d p r o c e d u r e o f Sharma and Wang ( 1 9 7 9 ) . C a l m o d u l in , i n t h e p r e s e n c e o f c a l c i u m , i n c r e a s e s PDE a c t i v i t y q u a n t i t a t i v e l y . In t h e a b s e n c e o f c a l c i u m ( i n t h e p r e s e n c e o f EGTA), CaM n e i t h e r s t i m u l a t e s n o r b i n d s t o p h o s p h o d i e s t e r a s e . Only t h e PDE i s l i m i t i n g i n t h e a s s a y .

137

A s s a y f o r C a l m o d u l i n a c t i v i t y

cAMP ■> 5 ' AMP -> A

( - ) EGTA

P h o s p h o d i e s t e r a s e <— ( + ) — C a l m o d u l i n (EC 3 . 1 . 4 . 1 7 )

5 ' N u c l e o t i d a s e (EC 3 . 1 . 3 . 5 )

( + ) Ca+2

F igu re 24.

139

v o r t e x i n g . P r e c i p i t a b l e m a t e r i a l was removed by c e n t r i f u g a t i o n f o r 10

min a t 1 0 ,0 0 0 x g . Two a l i q u o t s o f 0 . 4 ml e a c h were removed and added

t o 100 p i w a t e r and 100 p i o f ammonium m o ly b d a te r e a g e n t ( 2 . 5 g / 1 0 0 ml

o f 1 .2 5 N s u l f u r i c a c i d ) . The d e t e r m i n a t i o n o f i n o r g a n i c p h o s p h a t e

was i n i t i a t e d by a d d i n g t h e F i s k e - S u b a r r o w r e a g e n t ( 0 . 7 9 4 g / 1 0 ml

w a t e r ) and i n c u b a t i n g t h e sample a l i q u o t s a t room t e m p e r a t u r e f o r 10

m i n u t e s . The a b s o r p t i o n o f t h e sample was m e a s u r e d a t 660 nm u s i n g a

Beckman model 25, U V / v i s i b l e d u a l beam s p e c t r o p h o t o m e t e r . Each a s s a y

s e t i n c l u d e d a b l a n k c o n t a i n i n g a l l r e a g e n t s e x c e p t c a l m o d u l i n . The

amount o f PDE u t i l i z e d i n t h e a s s a y (4 mU/10 p l / s a m p l e ) was l e s s t h a n

t h a t u s e d by Sharma and Wang (12 - 15 mU) w i t h c o m p a r a b le r e d u c t i o n o f

o t h e r r e a g e n t v o lu m e s . Components o f t h e F i s k e - S u b a r r o w r e a g e n t were

s u p p l i e d p r e - m i x e d and were u s e d t o p r e p a r e a que ous r e a g e n t

i m m e d i a t e l y b e f o r e e a c h u s e ( 0 . 7 9 4 g / 1 0 ml w a t e r ) . The i n c u b a t i o n

t im e s were t h e same a s d e s c r i b e d by Sharma and Wang.

P r o t e i n c o n t e n t was d e t e r m i n e d u s i n g b o t h t h e UV method o f

W addel l ( 1956) and a m o d i f i c a t i o n o f t h e B r a d f o r d (1976) p r o c e d u r e .

The B r a d f o r d r e a g e n t was u s e d a t a c o n c e n t r a t i o n f i v e f o l d h i g h e r t h a n

o r i g i n a l l y r e p o r t e d by B r a d f o r d and was mixed d i r e c t l y w i t h t h e sample

( 0 . 2 ml B r a d f o r d 5x r e a g e n t and 0 . 8 ml s a m p l e ) . A l l a s s a y s were

p e r f o r m e d w i t h a t l e a s t 3 r e p l i c a t e s and a t l e a s t one p r o t e i n b l a n k .

P r o t e i n c o n t e n t i s e x p r e s s e d as BSA e q u i v a l e n t s (S igma #A9647,

f r a c t i o n V, l o t #10F0141) .

140

The Bradford reagent at 5 fold concentration (100 mg Coomassie

Brilliant Blue G-250, 50 ml ethanol, 100 ml 8 5 .5 % phosphoric acid,

50 ml w a t e r ) i s c o n s i d e r a b l y more s t a b l e i n s t o r a g e t h a n t h e o r i g i n a l

r e a g e n t r e p o r t e d by B r a d f o r d . However , t h e p r o t e i n - d y e complex i s

l e s s s t a b l e so t h e sam p les s h o u l d be m easu red i m m e d i a t e l y a f t e r

p r e p a r a t i o n . R e p e a t m easu rem en ts were p e r f o r m e d w i t h i n 20 min o f

m ix in g .

Calculation of Calmodulin Unit Activity.

D e t e r m i n a t i o n o f t h e u n i t a c t i v i t y o f s p i n a c h and m aize

c a l m o d u l i n was b a s e d upon t h e u n i t d e f i n i t i o n o f Sharma and Wang

(1979) f o r 0 .0 1 2 u n i t s o f p h o s p h o d i e s t e r a s e . One u n i t o f c a l m o d u l i n

has b e e n d e f i n e d as t h a t amount o f c a l m o d u l i n which y i e l d s a 50 %

maximal s t i m u l a t i o n o f 0 . 0 1 2 u n i t s o f p h o s p h o d i e s t e r a s e i n 1 ml o f

a s s a y medium above t h e b a s a l a c t i v i t y o f PDE, i n t h e p r e s e n c e o f

c a l c i u m . T h i s a c t i v i t y must be c a l c i u m d e p e n d e n t . T h a t i s , t h e

s t i m u l a t i o n a t t r i b u t e d t o c a l m o d u l i n must be r e v e r s i b l e t o t h e b a s a l

l e v e l by s u b s t i t u t i o n o f EGTA f o r c a l c i u m a t e q u i v a l e n t c o n c e n t r a t i o n .

S t a n d a r d c u r v e s were g e n e r a t e d f o r e a ch s o u r c e o f CaM. The s t a n d a r d

c u r v e s were p l o t t e d as p e r c e n t a c t i v a t i o n o f t h e b a s a l , c o n t r o l l e v e l

( t a k e n a s 100 %) a g a i n s t t h e e q u i v a l e n t volume o f sample added t o t h e

a s s a y medium. T o t a l volume d i d n o t e x c e e d 20 p i , and e q u i v a l e n t

vo lumes were p r e p a r e d by d i l u t i o n o f s a m p le . The p r e c i s i o n o f t h e

141

m easu rem en ts was m a i n t a i n e d by u s i n g a t o t a l sample volume o f 10 p i f o r

a l l s a m p le s e x c e p t s a t u r a t i n g l e v e l s o f sample (20 p i ) . The s t a n d a r d

c u r v e s p r e s e n t e d l a t e r ( F i g . 28) a r e c a l c u l a t e d c u r v e s .

A s s e s s m e n t o f Sample P u r i t y .

CaM e x t r a c t i o n s were r o u t i n e l y m ea s u red f o r h o m o g e n e i ty u s i n g

SDS-PAGE on 12 %, and 6 - 20 % l i n e a r g r a d i e n t g e l s u t i l i z i n g t h e g e l

f o r m u l a t i o n o f Laemmli (1970 , and Append ix B ) . Gels were s t a i n e d

u s i n g t h e s i l v e r s t a i n t e c h n i q u e o f M e r r i l e t a l . (1981 ) w i t h a

m o d i f i c a t i o n o f t h e f i x and wash p r o t o c o l s i m i l a r t o t h a t recommended

by Bio-Rad L a b o r a t o r i e s ( b u l l e t i n # 1 0 9 8 ) .

C a lm o d u l in does n o t s t a i n w e l l u s i n g t h e s i l v e r s t a i n p r o c e d u r e s .

However , r e s t a i n i n g ( d o u b le s t a i n i n g ) o f t h e g e l s a f t e r c o m p l e t e

d e s t a i n i n g , b r i n g s ou t t h e CaM bands i n t e n s e l y . Gels were r o u t i n e l y

d e s t a i n e d by a s o l u t i o n c o n t a i n i n g 6 g / 1 p o t a s s i u m d i c h r o m a t e and

2 m l / 1 s u l f u r i c a c i d (97 %). D e s t a i n i n g was a r r e s t e d by u s i n g a

s o l u t i o n o f 30 g / 1 sod ium s u l f i t e . G e ls were t h o r o u g h l y washed b e f o r e

r e s t a i n i n g . As l i t t l e as 0 .2 5 ng p r o t e i n was d e t e c t a b l e u s i n g d o u b le

s t a i n i n g w i t h t h e m o d i f i e d M e r r i l p r o c e d u r e .

142

P r o c e s s i n g o f t h e P h e n o t h i a z i n e - A f f i g e l Column.

The final purification step utilizes a phenothiazine affinity

gel. The use of affinity columns for CaM purification has been

reported previously for both animal (Jamieson and Vanaman, 1979) and

plant (Charbonneau and Cormier, 1979) systems. However, the affinity

gels used in these studies were prepared by the individual researchers

using phenothiazine derivatives which are not readily available. The

gel used in this study is available commercially and when used with

the modified application and elution buffers described here, its

performance is comparable to that of the gels reported previously.

The b u f f e r s f o r a p p l i c a t i o n and e l u t i o n o f c a l m o d u l i n f rom

p h e n o t h i i a z i n e - A f f i g e l (P-A) columns (B io -R ad b u l l e t i n #1088) were

m o d i f i e d t o i n c l u d e h i g h e r i o n i c s t r e n g t h s o f c a l c i u m . I n a d d i t i o n ,

EDTA was s u b s t i t u t e d f o r EGTA. The P-A column ( 0 . 7 x 4 . 5 cm) was

e q u i l i b r a t e d w i t h a t l e a s t 10 bed vo lumes o f P-A a p p l i c a t i o n b u f f e r

(300 mM NaCl, 1 mM C a C ^ , 50 mM T r i s - b a s e , 1 mM I3-MCE, 0 .0 1 % sod ium

a z i d e , pH 7 . 5 ) . E l u t i o n o f sample w i t h P-A e l u t i o n b u f f e r (20 mM

Na2EDTA, 50 mM T r i s - b a s e , 0 .0 1 % s od ium a z i d e , pH 7 . 5 ) was c o n t i n u e d

u n t i l no d e t e c t a b l e p r o t e i n was p r e s e n t .

A f t e r u s e , t h e column was c l e a n e d w i t h 20 bed vo lumes o f 8 M

p r e - c l e a n e d u r e a i n P-A a p p l i c a t i o n b u f f e r . U re a was c l e a n e d by

p a s s i n g i t t h r o u g h an AG-501x8 mixed bed g e l (B io -R ad ) column ( 1 . 5 x

40 cm) t w i c e . A f t e r c l e a n i n g w i t h u r e a , t h e column was

r e - e q u i l i b r a t e d w i t h P-A a p p l i c a t i o n b u f f e r f o r s u b s e q u e n t u s e .

143

E x t r a c t i o n o f C a l m o d u l i n .

A l l s t e p s were p e r f o r m e d on i c e o r i n a c o l d room (4 C) . A p i c a l

segm en ts o f m aize r o o t t i s s u e ( 3 0 0 g ) , 1 . 5 - 2 . 0 cm i n l e n g t h , were

hom ogen ized i n an e q u i v a l e n t volume o f e x t r a c t i o n b u f f e r . The

homogenate was f i l t e r e d t h r o u g h 50 pm n y l o n mesh and t h e b r e i was

r e - h o m o g e n i z e d i n an a d d i t i o n a l one h a l f volume o f e x t r a c t i o n b u f f e r .

The f i l t r a t e s were combined and c e n t r i f u g e d a t 15 ,000 x g f o r 30 min

( S o r v a l l RC2 - B c e n t r i f u g e e q u ip p e d w i t h a t y p e SS - 34 r o t o r ) . The

s u p e r n a t a n t was b r o u g h t t o 40 % s a t u r a t i o n w i t h g r a n u l a t e d ammonium

s u l f a t e ( 2 8 . 2 g / 1 0 0 g o f s u p e r n a t a n t ) . A f t e r e q u i l i b r a t i n g w i t h t h e

ammonium s u l f a t e f o r 30 min from t h e end o f ammonium s u l f a t e a d d i t i o n ,

t h e s u p e r n a t a n t was c e n t r i f u g e d a t 20 ,000 x g f o r 30 m i n u t e s . The

p e l l e t was d i s c a r d e d and t h e s u p e r n a t a n t b r o u g h t t o 70 % s a t u r a t i o n i n

ammonium s u l f a t e ( 1 5 .7 2 g / 1 0 0 g s u p e r n a t a n t ) , e q u i l i b r a t e d f o r 30 min ,

and c e n t r i f u g e d a t 20 ,000 x g f o r 30 m i n u t e s . The p e l l e t t e d m a t e r i a l ,

r e p r e s e n t i n g t h e 40 - 70 % ammonium s u l f a t e p r e c i p i t a b l e f r a c t i o n , was

r e s u s p e n d e d in a minimum volume o f P-A a p p l i c a t i o n b u f f e r and d i a l y z e d

a g a i n s t P-A a p p l i c a t i o n b u f f e r f o r 24 h w i t h one b u f f e r change and a

sample t o b u f f e r r a t i o o f 1 /1 0 0 .

144

The d i a l y z e d , m a t e r i a l was t h e n t r a n s f e r r e d t o a f l a s k o f tw i c e

t h e sample volume (100 m l) and h e a t t r e a t e d by immers ing t h e sample in

a b o i l i n g w a t e r b a t h . The sample was s t i r r e d r a p i d l y w h i l e m o n i t o r i n g

t h e t e m p e r a t u r e . When t h e t e m p e r a t u r e o f t h e sample r e a c h e d 80 C, i t

was i m m e d i a t e l y t r a n s f e r r e d t o an i c e b a t h w i t h r a p i d s t i r r i n g , and

a l l o w e d t o c o o l f o r a t l e a s t 1 h o r u n t i l t h e t e m p e r a t u r e was be low

10 C. The d e n a t u r e d p r o t e i n was removed by c e n t r i f u g i n g t h e sample a t

20 ,000 x g f o r 1 h . The s u p e r n a t a n t was t h e n a p p l i e d a t a f l o w r a t e

o f 0 . 8 m l /m in t o t h e p h e n o t h i a z i n e - A f f i g e l column ( 0 . 7 x 4 . 5 cm) which

was e q u i l i b r a t e d p r e v i o u s l y w i t h P-A a p p l i c a t - i o n b u f f e r . The f i r s t

e l u a t e was r e - a p p l i e d t o t h e colum n. The column was t h e n washed w i t h

P-A a p p l i c a t i o n b u f f e r u n t i l t h e 280 nm a b s o r b a n c e d e c l i n e d t o w i t h i n

0 .0 5 O°280 o f t h e i n i t i a l , p r e - l o a d b a s e l i n e . The column was t h e n

e l u t e d w i t h P-A e l u t i o n b u f f e r w h i l e m o n i t o r i n g a b s o r b a n c e . Samples

were c o l l e c t e d i n f o u r f i n a l f r a c t i o n s o f 4 0 , 40 , 40, and 100 ml and

l a b e l l e d as CaM # 1 - CaM # 4 . I n o r d e r t o remove EDTA and t o

s t a b i l i z e t h e c a l m o d u l i n , t h e f r a c t i o n s were i m m e d i a t e l y d i a l y z e d f o r

24 h ( s a m p le t o b u f f e r r a t i o o f 1 / 2 0 ) a g a i n s t P-A a p p l i c a t i o n b u f f e r

p r e p a r e d w i t h o u t NaCl. The b u f f e r was c hanged two t i m e s .

The d i a l y z e d sam ples were a s s a y e d f o r p r o t e i n and c a l m o d u l i n

c o n t e n t . Homogenei ty o f t h e s a m p le s was d e t e r m i n e d by SDS-PAGE.

Those s a m p le s c o n t a i n i n g c a l m o d u l i n were d i a l y z e d o v e r n i g h t ( two

ch anges o f b u f f e r ) a g a i n s t d o u b l e d i s t i l l e d w a t e r a d j u s t e d t o pH 7 .5

w i t h 10 f o l d c o n c e n t r a t e d P-A a p p l i c a t i o n b u f f e r p r e p a r e d w i t h o u t

145

NaCl. The d i a l y z e d s a m p le s were l y o p h y l i z e d t o d r y n e s s and

r e s u s p e n d e d in a minimum volume o f PDE b u f f e r .

T h i s same p r o c e d u r e was u s e d f o r e x t r a c t i o n and p u r i f i c a t i o n o f

CaM f rom d e b l a d e d s p i n a c h l e a v e s (1500 g ) . The c o m p l e t e e x t r a c t i o n ,

p u r i f i c a t i o n and q u a n t i f i c a t i o n , i n c l u d i n g d i a l y s i s r e q u i r e s l e s s t h a n

96 h and u s u a l l y can be c o m p l e t e d i n 72 h o u r s .

Results and Discussion.

C a lm o d u l in d i d n o t s t a i n w e l l u s i n g t h e M e r r i l s i l v e r s t a i n

p r o c e d u r e . P o l y a c r y l a m i d e g e l s c o n t a i n i n g SDS f o r m o l e c u l a r w e i g h t

d e t e r m i n a t i o n , when s t a i n e d a c c o r d i n g t o t h e p u b l i s h e d p r o c e d u r e o f

M e r r i l e t a l . ( 1981) o r a c c o r d i n g t o t h e m o d i f i e d p r o c e d u r e

recommended by B io -Rad L a b o r a t o r i e s ( b u l l e t i n # 1 0 9 8 ) , y i e l d e d a c l e a r

s p o t ( w i t h o r w i t h o u t a h a l o ) f o r t h e c a l m o d u l i n s a m p l e s . The c l e a r

s p o t which i n d i c a t e s t h e p r e s e n c e o f c a l m o d u l i n was c o n s i d e r a b l y

l i g h t e r t h a n t h e b a c k g ro u n d o f t h e g e l ( P l a t e V I I ) . When t h e g e l was

d e s t a i n e d and r e s t a i n e d as d e s c r i b e d i n M a t e r i a l s and M eth ods , t h e

c a l m o d u l i n band became q u i t e d i s t i n c t . R e s t a i n i n g was done from t h e

i n i t i a l s t e p ( t r e a t i n g t h e g e l w i t h t h e o x i d i z e r s o l u t i o n ) , and n o t as

s u g g e s t e d b y t h e Bio -Rad L a b o r a t o r i e s b u l l e t i n ( t r e a t i n g w i t h t h e

s i l v e r n i t r a t e s o l u t i o n ) . Double s t a i n i n g ( s t a i n , d e s t a i n , s t a i n ) o f

P l a t e V II .

Com par i son o f M a ize , S p i n a c h and Bov ine C a l m o d u l in Us ing SDS-PAGE.

The sam p les were v i s u a l i z e d by t h e m o d i f i e d M e r r i l s i l v e r s t a i n p r o c e d u r e u s i n g SDS-PAGE. The g e l (12%) was s t a i n e d one t ime a c c o r d i n g t o t h e m o d i f i e d m e thod . The bands a t t h e a r ro w s a r e c a l m o d u l i n . The c a l m o d u l i n does n o t s t a i n and i s even l i g h t e r t h a n t h e b a c kg round s t a i n i n g o f t h e g e l . The c a l m o d u l i n bands became q u i t e d i s t i n c t a f t e r d o u b l e s t a i n i n g ( c f . P l a t e s V I I , V I I I ) . S e v e r a l c o n t a m i n a t i n g p r o t e i n s were o b s e r v e d t o c o - e l u t e w i t h m aize c a l m o d u l i n f rom t h e a f f i n i t y co lumn. A s e c o n d a f f i n i t y p u r i f i c a t i o n o f m aize c a l m o d u l i n sample removed most o f t h e s e p r o t e i n s . A l l s am p les were p r e p a r e d i n Laemmli b u f f e r c o n t a i n i n g 0 . 1 mM CaCl^* Lane 1 ,6 :M o l e c u l a r w e i g h t s t a n d a r d s ; 2: Maize c a l m o d u l i n a f t e r a s eco n da f f i n i t y p u r i f i c a t i o n ; 3: Maize c a l m o d u l i n a f t e r a s i n g l e a f f i n i t yp u r i f i c a t i o n ; 4: Bovine c a l m o d u l i n ; 5: S p i n a c h c a l m o d u l i n a f t e r as i n g l e a f f i n i t y p u r i f i c a t i o n ; 7: Maize CaM sample p r i o r t o a f f i n i t yp u r i f i c a t i o n . A l l s a m p le s were a p p l i e d a t c a . 30 ng t o t a l p r o t e i n .The MM s t a n d a r d s were a p p l i e d a t 5 n g / e a c h p r o t e i n s t a n d a r d . The sam ple volume was 10 p i e x c e p t f o r t h e b o v i n e CaM (5 p i ) . The s t a n d a r d s were lysozyme ( 1 4 . 4 k d ) , s o y b e a n t r y p s i n i n h i b i t o r ( 2 1 . 5 k d ) , c a r b o n i c a n h y d r a s e ( 3 1 . 0 k d ) , o va lbum in ( 4 5 . 0 k d ) , b o v i n e se rum a lb u m in ( 6 6 . 2 k d ) , and p h o s p h o r y l a s e B ( 9 2 . 5 k d ) . The MU s t a n d a r d s a r e i n d i c a t e d by t h e b a r s t o t h e l e f t o f l a n e 1. The a r ro w s t o t h e r i g h t o f l a n e s 2 - 5 i n d i c a t e t h e CaM w hich i s v i s i b l e as a c l e a r s p o t . The b a r s t o t h e r i g h t o f l a n e 3 i n d i c a t e t h e c o n t a m i n a t i n g p r o t e i n s p r e s e n t a f t e r a s i n g l e a f f i n i t y p u r i f i c a t i o n .

146

147

P l a t e V I I .

1 2 3 4 5 6 7

148

e i t h e r 6 - 20 % l i n e a r g r a d i e n t o r 12 % g e l s y i e l d e d d i s t i n c t

c a l m o d u l i n bands ( P l a t e V I I I ) . Coomassie s t a i n i n g was n o t as

s e n s i t i v e as s i l v e r s t a i n i n g and i s n o t recommended u n l e s s t h e sample

i s known t o c o n t a i n h i g h c o n c e n t r a t i o n s o f c a l m o d u l i n . The s i l v e r

s t a i n i n g method r e v e a l e d c o n t a m i n a t i n g p r o t e i n s n o t v i s i b l e w i t h

Coomassie s t a i n t e c h n i q u e s .

I s o l a t i o n and p u r f i c a t i o n o f c a l m o d u l i n f rom m aize r o o t t i s s u e

y i e l d e d a p p r o x i m a t e l y a 200 f o l d c o n c e n t r a t i o n w i t h 43 % r e c o v e r y

b a s e d upon t o t a l i n i t i a l a c t i v i t y ( T a b l e 9) and more t h a n 80 %

r e c o v e r y from t h e ammonium s u l f a t e p r e c i p i t a t e d f r a c t i o n . The t o t a l

s t i m u l a t i o n o f PDE by t h e i n i t i a l hom ogena te ( S u p e r n a t a n t 1) was n o t

e n t i r e l y r e v e r s i b l e by s u b s t i t u t i o n o f EGTA f o r c a l c i u m i n t h e PDE

a s s a y . The i n i t i a l homogenate s t i m u l a t e d PDE a c t i v i t y by 4 1 3 .7 %/10 p i

sa m p le . However, i n t h e p r e s e n c e o f EGTA, s u b s t a n t i a l s t i m u l a t i o n

( 1 9 4 . 2 %/10 p i ) p e r s i s t e d . T h u s , t h e c a l c i u m d e p e n d e n t s t i m u l a t i o n o f

PDE a c t i v i t y was 320 - 322 % (EGTA a d j u s t e d A ^ q v a l u e , 415 - 95 %).

Based on t h e f i n a l p r o t e i n c o n t e n t o f t h e p u r i f i e d sa m p le , ab o u t

0 .3 7 % o f t h e t o t a l p r o t e i n e x t r a c t a b l e f rom r o o t s i s c a l m o d u l i n .

However , e x a m i n a t i o n o f t h e p u r i f i e d sample on SDS-PAGE, u s i n g t h e

m o d i f i e d M e r r i l s i l v e r s t a i n , r e v e a l e d t h e p r e s e n c e o f s e v e r a l

c o n t a m i n a t i n g p r o t e i n s ( P l a t e V I I ) . These p r o t e i n s may have c a u s e d an

ov e r e s t i m a t e o f t o t a l p r o t e i n i n t h e p u r i f i e d sa m p le , even th ough th e

m o d i f i e d B r a d f o r d p r o t e i n a s s a y e x h i b i t s d e c r e a s e d s e n s i t i v i t y f o r

p r o t e i n s , such as c a l m o d u l i n , wh ich c o n t a i n a low p e r c e n t a g e o f

P l a t e V I I I .

Maize Calmodulin Purified on a Phenothiazine-Affinity Column Compared with the Sample Loaded onto the Column.

The s am p le l o a d e d was p a r t i a l l y p u r i f i e d by ammonium s u l f a t e f r a c t i o n a t i o n and h e a t t r e a t m e n t as d e s c r i b e d i n t h e M a t e r i a l s and M ethods . The g e l (6 - 20% g r a d i e n t ) was r e s t a i n e d u s i n g t h e m o d i f i e d M e r r i l p r o c e d u r e . The sample l o a d e d o n t o t h e a f f i n i t y column c o n t a i n e d numerous p r o t e i n s which a r e r e s i s t a n t t o h e a t t r e a t m e n t . C a l m o d u l in i s p r e s e n t i n t h e f i r s t f r a c t i o n o f t h e column e l u t i o n ( l a n e 3 ) , w i t h o u t any d e t e c t a b l e CaM i n t h e r e m a i n i n g e l u t i o n f r a c t i o n s ( l a n e s 3 - 6 ) . A l l s am p les were p r e p a r e d i n Laemmli b u f f e r c o n t a i n i n g 0 . 1 mM CaCl^* Sample vo lumes and p r o t e i n c o n t e n t were as i n P l a t e V I I . Lane 1: M o l e c u l a r w e i g h t s t a n d a r d s ; 2: Maize CaM,p a r t i a l l y p u r i f i e d sample a p p l i e d t o t h e a f f i n i t y co lumn. Sample was ammonium s u l f a t e f r a c t i o n a t e d (40 - 70%) and h e a t t r e a t e d ; 3: M a ize ,f i r s t a f f i n i t y e l u t i o n , f i r s t e l u t i o n f r a c t i o n (CAM # 1 ) ; 4: BovineCaM; 5: M a ize , f i r s t a f f i n i t y e l u t i o n , s e cond f r a c t i o n (CAM # 2 ) ; 6:M a iz e , f i r s t a f f i n i t y e l u t i o n , t h i r d f r a c t i o n (CAM # 3 ) . S e v e r a l c o n t a m i n a t i n g p r o t e i n s a r e p r e s e n t a f t e r a s i n g l e a f f i n i t y p u r i f i c a t i o n o f m a iz e CaM. T h e s e c o n t a m i n a n t s a r e v i s i b l e i n l a n e 3 b e tw e e n 2 1 .5 - 3 1 . 0 kd and 4 5 . 0 - 6 6 . 2 kd . No CaM was d e t e c t e d in f r a c t i o n s CaM # 2, 3.

149

150

P l a t e V I I I .

92.566.2

45.031.0

2 1.5 14.4

1 2 3 4 5 6

T ab le 9 .

C om par i son o f S p e c i f i c and T o t a l C a l m o d u l in A c t i v i t y D ur ing P r o g r e s s i v e P u r i f i c a t i o n f rom Maize R o o t s and S p i n a c h L e a v e s .

P r o t e i n c o n t e n t was d e t e r m i n e d by t h e m o d i f i e d method o f B r a d f o r d and by t h e method o f W adde l l ( 1 9 5 6 ) . U n i t s o f c a l m o d u l i n a c t i v i t y w e re c a l c u l a t e d from s t a n d a r d c u r v e s f o r e a c h s o u r c e o f c a l m o d u l i n . R e c o v e r y o f a c t i v i t y was d e t e r m i n e d from t h e t o t a l c a l c u l a t e d a c t i v i t y p r e s e n t i n t h e s a m p l e s . U n i t s o f a c t i v i t y a r e b a s e d upon t h e u n i t d e f i n i t i o n d e s c r i b e d i n t h e R e s u l t s .

151

152

T ab le 9.

Sample Protein mg/ml

CaMU/ml

CaM T o t a l A c t i v i t yU/mg_____________ U n i t s _____

HomogenateMaizeS p i n a c h

40-70%ammoniums u l f a t ef r a c t i o n

S p i n a c h

Heat t r e a t e d Maize S p i n a c h

Flow t h r o u g h o f a f f i n i t y column

1.022 .0 3

19 .89

1 .821 .8 9

58134

207

120300

5766.1

1 0 .4

46159

96 ,4 1 027 8 ,184

26 ,4 3 2

4 7 ,2 8 0

MaizeS p i n a c h

CaM #1 Maize S p i n a c h

CaM # 1 , 2nd A f f i n i t y e l u t i o n

Maize

1 .171 .85

0 .0 70.02

4450

873500

3 . 4243

0 .0 3 810

12 ,68025 ,1 26

27 ,375

4 1 ,9 0 421,000

40 ,2 41

153

a r o m a t i c amino a c i d s . Measurement o f p r o t e i n c o n t e n t by t h e UV method

o f W addel l y i e l d e d s i m i l a r v a l u e s . When t h e a f f i n i t y - p u r i f i e d

c a l m o d u l i n sample o b t a i n e d from m a iz e was r e - l o a d e d o n t o t h e

p h e n o t h i a z i n e - a f f i n i t y co lumn, t h e c o n t a m i n a t i n g p r o t e i n s a p p a r e n t l y

were n o t r e t a i n e d ( P l a t e V I I , I X ) . The m ea s u red p r o t e i n c o n t e n t o f

t h e r e - p u r i f i e d sample was low er ( c a . 0 . 0 3 mg/m l) w i t h o u t a p p r e c i a b l e

d e c r e a s e i n c a l m o d u l i n . T h i s s u g g e s t s t h a t t h e c a l m o d u l i n c o n t e n t o f

m a ize r o o t t i s s u e i s b e tw e e n 0 . 3 0 and 0 .3 7 %. The low er l i m i t i s

o b t a i n e d from t h e seco n d a f f i n i t y p u r i f i e d s a m p l e . The u p p e r l i m i t i s

c a l c u l a t e d from t h e f i r s t a f f i n i t y p u r i f i c a t i o n and p resum es t h a t t h e

sample was p u r e c a l m o d u l i n . T h a t t h e c o n t a m i n a t i n g p r o t e i n s do n o t

b i n d t o t h e a f f i n i t y g e l when a p p l i e d i n a s a m p le p r e d o m i n a t e d by

c a l m o d u l i n s u g g e s t s t h a t t h e s e p r o t e i n s may be b i n d i n g e i t h e r

n o n - s p e c i f i c a l l y o r , more p r o b a b l y , w i t h l ow e r s p e c i f i t y t h a n

c a l m o d u l i n t o t h e p h e n o t h i a z i n e a f f i n i t y g e l . T h e s e p r o t e i n s may

r e p r e s e n t a d d i t i o n a l c a l c i u m m o d u l a t o r p r o t e i n s .

I n e x t r a c t s o f b o t h s p i n a c h and m a i z e , no d e t e c t a b l e c a l m o d u l i n

was found i n any a f f i n i t y column e l u t i o n sam ple o t h e r t h a n t h e f i r s t

sample ( P l a t e V I I ) . A c o m p a r i s o n o f SDS-PAGE g r a d i e n t g e l s o f

a f f i n i t y p u r i f i e d sam p les ( P l a t e V I I I , X, X I ) d e m o n s t r a t e s t h e

e f f e c t i v e n e s s o f t h e p h e n o t h i a z i n e g e l . A l t h o u g h a l a r g e p e r c e n t a g e

o f t h e t o t a l e x t r a c t a b l e p r o t e i n i s d e n a t u r e d and p r e c i p i t a t e d by t h e

h e a t t r e a t m e n t , numerous p r o t e i n s , i n c l u d i n g t h e c a l m o d u l i n , a r e h e a t

s t a b l e .

P l a t e IX.

The E l e c t r o p h o r e t i c M o b i l i t y o f Maize , S p i n a c h and Bov ine C a l m o d u l i n .

The m a iz e c a l m o d u l i n s am p le shown i n l a n e 2 was p u r i f i e d on t h ea f f i n i t y column t w i c e . V i r t u a l l y a l l o f t h e p r o t e i n s which c o - e l u t e d d u r i n g t h e f i r s t a f f i n i t y p u r i f i c a t i o n a p p a r e n t l y were removed d u r i n g t h e s e c o n d a f f i n i t y p u r i f i c a t i o n ( c f . l a n e 2 , 3 ; a l s o s e e P l a t e V I I ) . The m o l e c u l a r w e i g h t s o f b o t h m a iz e r o o t and s p i n a c h l e a f CaM a p p e a r t o be low er t h a n t h a t o f b o v i n e CaM. The m o l e c u l a r w e i g h t o f t h e m a iz e c a l m o d u l i n was low er t h a n t h a t o f s p i n a c h c a l m o d u l i n . The MW o f s p i n a c h and b o v i n e c a l m o d u l i n a p p e a r e d t o be c l o s e r t h a n t h a t o f m aize and s p i n a c h CaM. Lane 1 , 6 : MW s t a n d a r d s , as i n P l a t e V I I ; 2: MaizeCaM, s e c o n d a f f i n i t y p u r i f i c a t i o n ; 3: Maize CaM, f i r s t a f f i n i t yp u r i f i c a t i o n ; 4: Bovine CaM; 5: S p in a c h CaM, f i r s t a f f i n i t y p u r i f i c a t i o n . A l l s am p les w ere p r e p a r e d i n Laemmli sam ple b u f f e r c o n t a i n i n g 0 . 1 mM C a C ^ * The MW s t a n d a r d s a r e i n d i c a t e d by b a r s t ot h e l e f t o f l a n e 1 and t o t h e r i g h t o f l a n e 6.

154

155

P l a t e IX.

1 2 3 4 5 6

P l a t e X.

P r o g r e s s i v e P u r i f i c a t i o n o f S p in a c h L e a f C a lm o d u l in : S i n g l e S t a i n i n g .

A f f i n i t y p u r i f i e d s p in a c h CaM sam ple was i n i t i a l l y t h o u g h t n o t t o c o n t a i n t h e c o - e l u t i n g c o n ta m in a t in g p r o t e i n s o b s e rv e d i n t h e m aize r o o t sa m p le . However, a p p l i c a t i o n o f a l a r g e r (100 n g ) s p in a c h sam ple t o SDS-PAGE, 6 - 20% g r a d i e n t g e l s r e v e a l e d t h e p r e s e n c e o f t h e c o n t a m i n a t i n g p r o t e i n s i n a sam ple b e l i e v e d t o be homogeneous f o r s p in a c h c a lm o d u l in . T h e se p r o t e i n s a r e s i m i l a r i n m i g r a t i o n p a t t e r n t o t h o s e o b s e rv e d t o c o n ta m in a t e t h e m aize CaM sam ple ( c f . P l a t e s V I I , X I ) . The power o f t h e a f f i n i t y p u r i f i c a t i o n i s d e m o n s t r a te d by co m p ar in g t h e bands p r e s e n t a f t e r a f f i n i t y p u r i f i c a t i o n ( l a n e 5 ) w i th t h o s e p r e s e n t in th e p r i o r p u r i f i c a t i o n s t e p s ( l a n e s 2 - 4 ) .Lane 1 ,7 : MW s t a n d a r d s ; 2: S p in a c h hom ogenate a f t e r c e n t r i f u g a t i o nand f i l t e r i n g , d i l u t e d 1 /1 0 0 0 ; 3: Ammonium s u l f a t e f r a c t i o n a t i o n (40 - 70%) a f t e r d i a l y s i s ; 4: H ea t t r e a t e d sam ple a f t e r c e n t r i f u g a t i o n ; 5: A f f i n i t y colum n e l u t i o n (100 n g ) ; 6: BovineCaM. A l l sam p les w ere p r e p a r e d i n Laemmli sam p le b u f f e r c o n t a i n i n g 0 .1 mM CaCl^* P r o t e i n c o n t e n t was t h e same as i n P l a t e V I I , e x c e p t as n o t e d . The MW s t a n d a r d s a r e i n d i c a t e d by b a r s t o t h e l e f t o f l a n e 1 and t o t h e r i g h t o f l a n e 7 . The a r ro w s t o t h e r i g h t o f l a n e s 5 and 6 p o i n t t o t h e CaM bands w hich become v i s i b l e a f t e r d o u b le s t a i n i n g ( P l a t e X I ) . The b a r s t o t h e r i g h t o f l a n e 5 i n d i c a t e th e c o n t a m i n a t i n g p r o t e i n s p r e s e n t a f t e r a s i n g l e a f f i n i t y p u r i f i c a t i o n .

156

157

P l a t e X.

1

P l a t e XI.

P r o g r e s s i v e P u r i f i c a t i o n o f S p in a c h L e a f C a lm o d u l in : Double S t a i n i n g .

A f f i n i t y p u r i f i e d s p in a c h CaM sam ple was i n i t i a l l y th o u g h t n o t t o c o n t a i n t h e c o - e l u t i n g c o n ta m in a t in g p r o t e i n s o b s e rv e d in t h e m aize r o o t s a m p le . However, a p p l i c a t i o n o f a l a r g e r (100 ng ) s p in a c h sam ple t o SDS-PAGE, 6 - 20% g r a d i e n t g e l s r e v e a l e d th e p r e s e n c e o f t h e c o n ta m in a t in g p r o t e i n s in a sam p le b e l i e v e d t o be homogeneous f o r s p in a c h c a lm o d u l in . T h e se p r o t e i n s a r e s i m i l a r i n m i g r a t i o n p a t t e r n t o t h o s e o b s e rv e d t o c o n ta m in a t e t h e m a iz e CaM sam ple ( c f . P l a t e s V I I , X I ) . V i s u a l i z a t i o n o f t h e c a lm o d u l in n e c e s s i t a t e s d o u b le s t a i n i n g . A f t e r d o u b le d e s t a i n i n g , t h e CaM band i s q u i t e d i s t i n c t , b u t i t i s p r e s e n t as a c l e a r s p o t a f t e r o n ly s t a i n i n g once ( c f . P l a t e s X, X I ) . Lane 1 ,7 : MW s t a n d a r d s ; 2: S p in a c h hom ogenate a f t e rc e n t r i f u g a t i o n and f i l t e r i n g , d i l u t e d 1 /1 0 0 0 ; 3: Ammonium s u l f a t ef r a c t i o n a t i o n (40 - 70%) a f t e r d i a l y s i s ; 4: H e a t t r e a t e d sam plea f t e r c e n t r i f u g a t i o n ; 5: A f f i n i t y column e l u t i o n (100 n g ) ; 6:B ov ine CaM. A l l sa m p le s w ere p r e p a r e d i n Laemmli sam ple b u f f e r c o n t a i n i n g 0 .1 mM CaCl^* P r o t e i n c o n t e n t was t h e same as in P l a t e V I I , e x c e p t as n o t e d . The MW s t a n d a r d s a r e i n d i c a t e d by b a r s t o t h e l e f t o f l a n e 1 and t o t h e r i g h t o f l a n e 7. The a rro w s t o t h e r i g h t o f l a n e s 5 and 6 p o i n t t o t h e CaM bands w hich become v i s i b l e a f t e r d o u b le s t a i n i n g ( P l a t e X I ) . The b a r s t o t h e r i g h t o f l a n e 5 i n d i c a t e t h e c o n ta m in a t in g p r o t e i n s p r e s e n t a f t e r a s i n g l e a f f i n i t y p u r i f i c a t i o n .

158

159

160

The r e c o v e r y o f c a lm o d u l in from t h e s p in a c h e x t r a c t i o n was low

( a b o u t 6 %). In c o n t r a s t t o t h e m aize sa m p le , t h e r e w ere no

d e t e c t a b l e c o n ta m in a t in g p r o t e i n s p r e s e n t i n th e s p in a c h sam ple when

c o m p a ra b le p r o t e i n c o n c e n t r a t i o n s w ere exam ined ( P l a t e V I I ) . However,

i f c o n s i d e r a b l y h i g h e r c o n c e n t r a t i o n s o f s p in a c h c a lm o d u l in e l u t e d

from t h e a f f i n i t y g e l column w ere exam ined on SDS-PAGE, s e v e r a l

c o n ta m in a t in g p r o t e i n s w ere o b s e rv e d ( P l a t e X, X I ) . T hese p r o t e i n s

a r e s i m i l a r , i f n o t i d e n t i c a l , i n m o b i l i t y t o t h o s e o b s e rv e d t o

c o n ta m in a t e t h e m aize CaM s a m p le . T h is s u g g e s t s t h a t s e v e r a l p r o t e i n s

a r e p r e s e n t w i t h i n b o th m aize and s p in a c h t i s s u e w hich e x h i b i t b i n d in g

a f f i n i t i e s f o r p h e n o t h i a z i n e s i m i l a r t o t h a t o f c a lm o d u l in . Both th e

i n c r e a s e d p u r i t y o f t h e f i n a l sam ple and t h e low er r e c o v e r y o f t o t a l

c a lm o d u l in a c t i v i t y from s p in a c h l e a v e s as com pared w i th m aize r o o t s

may be a r e s u l t o f t h e u s e o f a much l a r g e r amount o f t i s s u e f o r t h e

s p in a c h l e a f e x t r a c t i o n (1500 g ) t h a n f o r t h e m aize r o o t e x t r a c t i o n

(300 g ) . C o n s id e r a b l e c a lm o d u l in a c t i v i t y r e m a in e d in t h e f lo w

th ro u g h ( e l u a t e ) o f t h e s p in a c h sam p le lo a d e d o n to t h e a f f i n i t y

colum n. T h is s u g g e s t s t h a t t h e b i n d i n g c a p a c i t y o f t h e g e l was

e x c ee d e d and t h a t th e volume o f g e l u s e d i n t h e column was i n a d e q u a te

t o b in d a l l o f t h e c a lm o d u l in p r e s e n t in t h e s p in a c h sa m p le . I f t h e

r e c o v e r y o f a c t i v i t y i s c a l c u l a t e d b a s e d on t o t a l a c t i v i t y p r e s e n t in

t h e ammonium s u l f a t e p r e c i p i t a t e d p r o t e i n , t h e n n e a r l y 80 % o f t h e

a c t i v i t y was r e c o v e r e d i n t h e a f f i n i t y p u r i f i e d s a m p le . I f r e c o v e r y

i s c a l c u l a t e d b a s e d on t o t a l a c t i v i t y i n t h e i n i t i a l e x t r a c t i o n , th e n

161

o n ly a b o u t 6 % o f t h e a c t i v i t y was r e c o v e r e d in t h e a f f i n i t y p u r i f i e d

s p in a c h sa m p le . T h is i n d i c a t e s a m a jo r l o s s o f a c t i v i t y d u r in g th e

ammonium s u l f a t e p r e c i p i t a t i o n . A p o r t i o n o f t h e l o s s in a c t i v i t y may

be a t t r i b u t e d t o t h e n o n - r e v e r s i b l e a c t i v i t y p r e s e n t in t h e i n i t i a l

e x t r a c t . However, o n ly a b o u t 10 - 15 % o f t h e t o t a l a c t i v i t y p r e s e n t

in t h e i n i t i a l hom ogenate i s r e c o v e r e d in t h e ammonium s u l f a t e

f r a c t i o n .

C a l c u l a t i o n o f t o t a l c a lm o d u l in i n t h e i n i t i a l hom ogena te , a f t e r

c o r r e c t i n g f o r t h e a c t i v i t y l o s t , s u g g e s t s t h a t t h e c a lm o d u l in

a c c o u n t s f o r a b o u t 0 .3 2 % o f t h e t o t a l p r o t e i n e x t r a c t a b l e from

s p in a c h l e a v e s . T h i s v a l u e i s in c l o s e a g re e m e n t w i th t h a t found f o r

t h e m aize r o o t t i s s u e and s i m i l a r t o t h a t o b s e rv e d f o r c a l f and r a t

b r a i n (Cheung, 1971; K a k iu c h i e t a l . , 1 9 7 3 ) . The p e r c e n ta g e o f t o t a l

p r o t e i n r e p r e s e n t e d by c a lm o d u l in i s low er th a n t h a t r e p o r t e d by

Slocum and Roux (1983) f o r o a t c o l e o p t i l e t i s s u e ( a t l e a s t 1 .0 %).

However, t h e y e s t i m a t e d c a lm o d u l in c o n t e n t by UV in d u ce d i r r e v e r s i b l e

b in d in g o f ^ C - c h l o r p r o m a z i n e , a m ethod w hich i s n o t h i g h l y s p e c i f i c ,

s i n c e c h lo r p r o m a z in e does n o t b in d w i t h h ig h s p e c i f i c i t y t o c a lm o d u l in

(W eiss e t a l . , 1 9 8 0 ) .

U n i t s o f c a lm o d u l in a c t i v i t y w ere d e te r m in e d f o r e a ch s o u r c e o f

c a lm o d u l in by g e n e r a t i n g s t a n d a r d c u r v e s from p u r i f i e d sam ples o f

m aize r o o t , s p in a c h l e a f , and c a l f b r a i n . The u n i t a c t i v i t y was

d e te r m in e d from a p l o t o f p e r c e n t a c t i v a t i o n ( c o n t r o l a s 100 %)

a g a i n s t e q u i v a l e n t vo lum es o f sam ple ( s a m p le s w ere d i l u t e d , w h i l e t h e

volume was h e l d c o n s t a n t ) and u s e d t o c a l c u l a t e t h e e q u i v a l e n t u n i t s

162

o f a c t i v i t y p r e s e n t i n 1 p i o f s a m p le . The o b s e rv e d s t i m u l a t i o n o f PDE

was t h e n r e p l o t t e d a g a i n s t t h e c a l c u l a t e d u n i t s o f a c t i v i t y p r e s e n t in

each sa m p le . The c a l c u l a t e d s t a n d a r d c u rv e s f o r e a c h s o u r c e o f

c a lm o d u l in a r e p r e s e n t e d in F i g u r e s 25 - 27 . The c o m p a r iso n o f

s t i m u l a t i o n o f PDE by c a lm o d u l in f o r e a c h o f th e t h r e e s o u r c e s o f

c a lm o d u l in ( F ig . 28) shows t h a t c a l f , s p i n a c h , and m aize c a lm o d u l in

a re s i m i l a r in t h e i r a b i l i t y t o a c t i v a t e b o v in e p h o s p h o d i e s t e r a s e .

T h is i s t o be e x p e c t e d f o r a p r o t e i n w hich e x h i b i t s a h ig h d e g r e e o f

c o n s e r v a t i o n in s t r u c t u r e be tw e en o r g a n i s m s . W a t te r s o n e t a l . (1980)

have r e p o r t e d s e v e r a l d i f f e r e n c e s b e tw e e n c a l f b r a i n and s p in a c h l e a f

c a lm o d u l in . Most n o t a b l e i s t h e a b s e n c e o f one t y r o s i n e and one

a r g i n y l r e s i d u e and a low er t h r e o n i n e / s e r i n e r a t i o i n s p in a c h . T hese

d i f f e r e n c e s may a c c o u n t f o r t h e low er m aximal s t i m u l a t i o n o f b o v in e

PDE by b o th s p in a c h l e a f and m aize r o o t c a lm o d u l in . W a t t e r s o n a l s o

r e p o r t e d t h a t s p in a c h l e a f c a lm o d u l in h a s a m o le c u la r w e ig h t low er

th a n t h a t o f c a l f b r a i n c a lm o d u l in . Based upon SDS-PAGE, t h i s i s th e

c a s e f o r b o th m aize r o o t and s p in a c h l e a f c a lm o d u l in ( P l a t e V I I I ) .

C a lm o d u l in from m aize r o o t a p p e a r s t o have a m o le c u la r w e ig h t low er

th a n t h a t o f s p in a c h l e a f c a lm o d u l in . However, t h e m o le c u la r w e ig h t

d i f f e r e n c e be tw e en s p in a c h l e a f and b o v in e b r a i n c a lm o d u l in does n o t

a p p e a r t o be as l a r g e as i n d i c a t e d by D i e t e r and Marme (1 9 8 2 ) . The

d i f f e r e n t i a l change i n e l e c t r o p h o r e t i c m o b i l i t y o f b o v in e , m a ize and

s p in a c h c a lm o d u l in s i n t h e p r e s e n c e o f c a lc iu m o r EDTA, r e p o r t e d by

Van E l d i k e t a l . ( 1 9 8 0 ) , was a l s o o b s e r v e d .

F ig u r e 25.

S ta n d a r d C urve f o r C a l f B r a in C a lm o d u l in

S t i m u l a t i o n o f PDE a c t i v i t y i s shown a s a p e r c e n t a g e s t i m u l a t i o n above t h e b a s a l (no CaM) a c t i v i t y o f PDE (% o f c o n t r o l ) . The b a s a l PDE a c t i v i t y was t a k e n as 100%. U n i t a c t i v i t y o f CaM was c a l c u l a t e d as d e s c r i b e d i n R e s u l t s . The c u r v e h a s b e e n c o r r e c t e d f o r EGTA i r r e v e r s i b l e s t i m u l a t i o n o f PDE a c t i v i t y .

163

% of

C

ontr

ol400

350_______

300 Bovine

250

20 0 IIItItI

150

100

IIIIfII

_l_0 -j 1--------- 1______ L.

4 6 Units of Calmodulin

F igu re 25.

F igu re 26.

S ta n d a r d C urve f o r S p in a c h L e a f C a lm o d u l in

S t i m u l a t i o n o f PDE a c t i v i t y i s shown as a p e r c e n ta g e s t i m u l a t i o n above t h e b a s a l (no CaM) a c t i v i t y o f PDE (% o f c o n t r o l ) . The b a s a l PDE a c t i v i t y was t a k e n as 100%. U n i t a c t i v i t y o f CaM was c a l c u l a t e d as d e s c r i b e d in R e s u l t s . The c u rv e h a s b e e n c o r r e c t e d f o r EGTA i r r e v e r s i b l e s t i m u l a t i o n o f PDE a c t i v i t y .

165

of C

ontr

ol400

350

300

S pinach

250

200

150

100

0 2 4 6 8 10U n i t s o f C a lm o d u l in

F ig u r e 26.O'O '

F ig u r e 27.

S t a n d a r d C urve f o r M aize Root C a lm o d u l in

S t i m u l a t i o n o f PDE a c t i v i t y i s shown as a p e r c e n t a g e s t i m u l a t i o n above t h e b a s a l (no CaM) a c t i v i t y o f PDE (% o f c o n t r o l ) . The b a s a l PDE a c t i v i t y was t a k e n as 100%. U n i t a c t i v i t y o f CaM was c a l c u l a t e d a s d e s c r i b e d i n R e s u l t s . The c u rv e has been c o r r e c t e d f o r EGTA i r r e v e r s i b l e s t i m u l a t i o n o f PDE a c t i v i t y .

167

% o

f C

ontr

ol400

350

300

Maize250

200

150

100

0 2 4 6 8 10Units o f C a lm o d u l in

F igu re 27.O'oo

F ig u r e 28.

C om parison o f S t i m u l a t i o n o f PDE by M aize , S p in a c h and Bovine C a lm o d u l in .

S t i m u l a t i o n o f PDE a c t i v i t y i s shown as a p e r c e n t a g e s t i m u l a t i o n above t h e b a s a l (no CaM) a c t i v i t y o f PDE. The b a s a l PDE a c t i v i t y was t a k e n a s 100%. A c t i v a t i o n o f PDE above t h e b a s a l ( c o n t r o l ) l e v e l was s i m i l a r f o r a l l t h r e e c a lm o d u l i n s . However, b o t h m aize r o o t and s p in a c h l e a f CaM r e a c h m axim al s t i m u l a t i o n a t a low er l e v e l th a n b o v in e CaM. U n i t a c t i v i t y f o r e a c h CaM was c a l c u l a t e d as d e s c r i b e d in R e s u l t s . The c u rv e s have b e e n c o r r e c t e d f o r EGTA i r r e v e r s i b l e s t i m u l a t i o n o f PDE a c t i v i t y .

169

% of

Con

trol

400

350

300

250

Bovine200

Maize

150 Spinach

100

0 2 4 6 8 10Units of C a lm odu l in

F ig u re 28.

171

I n summary, r a p i d i s o l a t i o n , p u r i f i c a t i o n and q u a n t i f i c a t i o n o f

c a lm o d u l in from t h r e e s o u r c e s was a c c o m p l i s h e d . C a lm o d u l in from a l l

t h r e e s o u r c e s e x h i b i t s p o o r s t a i n i n g w i th t h e m o d i f ie d M e r r i l s i l v e r

s t a i n u n l e s s t h e g e l s a r e d o u b le s t a i n e d . Both m aize r o o t and s p in a c h

l e a f c a lm o d u l in a p p e a r t o r e p r e s e n t a p p r o x i m a t e ly 0 .3 0 - 0 .4 0 % o f th e

e x t r a c t a b l e p r o t e i n . I f t h e t i s s u e sam ple i s a d j u s t e d f o r t h e s i z e o f

th e a f f i n i t y column ( a b o u t 100 g t i s s u e / 1 ml g e l ) , a t l e a s t 80 % o f

t h e t o t a l c a lm o d u l in can be i s o l a t e d . Both m aize and s p in a c h

c a lm o d u l in e x h i b i t a c t i v i t y s i m i l a r t o t h a t o f c a l f c a lm o d u l in w i th

r e g a r d t o t h e s t i m u l a t i o n o f p h o s p h o d i e s t e r a s e a c t i v i t y .

However, b o th o f t h e p l a n t c a lm o d u l in s a p p e a r t o have m o le c u la r

w e ig h ts low er t h a n c a l f b r a i n c a lm o d u l in . M aize r o o t c a lm o d u l in

a p p e a r s t o have a low er m o le c u la r w e ig h t t h a n s p in a c h l e a f c a lm o d u l in .

The p r e s e n c e o f a s m a l l number o f p r o t e i n s w hich i n i t i a l l y c o - e l u t e

w i th m a iz e r o o t and s p in a c h l e a f c a lm o d u l in from a p h e n o t h i a z i n e

a f f i n i t y column s u g g e s t s t h a t a d d i t i o n a l p r o t e i n s , p e rh a p s w i th

f u n c t i o n s s i m i l a r t o c a lm o d u l in a r e p r e s e n t in b o t h m aize r o o t and

s p in a c h l e a f t i s s u e .

i

SUMMARY

M aize r o o t t i s s u e in w hich t h e c e l l s have b e e n d i s r u p t e d e x h i b i t

i n c r e a s e d e x t e n s i o n u n d e r t e n s i o n when t r e a t e d w i th e i t h e r a c id

(pH 4 . 0 ) o r EDTA. The i n d u c t i o n o f e x t e n s i o n by EDTA c l o s e l y

r e s e m b le s a c i d - i n d u c e d e x t e n s i o n . A t t r i b u t e s o f b o th a l g a l (V a l o n i a )

and c o l e o p t i l e ( o a t ) c e l l w a l l l o o s e n i n g a p p e a r t o be p r e s e n t i n m aize

r o o t c e l l w a l l m a t e r i a l s i n c e F /T m aize r o o t s e x te n d d r a m a t i c a l l y in

r e s p o n s e to EDTA (a s does V a lo n ia b u t n o t o a t c o l e o p t i l e s ) b u t

a c i d - in d u c e d CWL i s a b o l i s h e d by d e n a t u r i n g t r e a t m e n t ( a s w i th o a t

c o l e o p t i l e s b u t n o t V a l o n i a ) . T h ese r e s u l t s s u g g e s t c e l l w a l l

lo o s e n in g i n m aize r o o t s i s e n z y m a t ic and may in v o lv e d i v a l e n t

c a t i o n s , p e rh a p s c a lc iu m , e i t h e r i n some c r o s s - l i n k i n g f u n c t i o n (a s in

V a l o n i a ) o r more p r o b a b ly as a m e d i a to r o f t h e e n z y m a tic CWL p r o c e s s .

Two r e s u l t s w hich a r e d i f f i c u l t t o r e s o l v e a r e t h e a p p a r e n t i n c r e a s e

i n p l a s t i c i t y a t 250 mg and t h e i n a b i l i t y o f EDTA t o i n c r e a s e

e x t e n s i b i l i t y in t h e a b s e n c e o f t e n s i o n . The i n c r e a s e in p l a s t i c i t y

in t h e p r e s e n c e o f v e r y l i t t l e t e n s i o n , a l t h o u g h anom olous , may

r e f l e c t a s e co n d c r i t i c a l s t r e s s l e v e l o f t h e c e l l w a l l m a t e r i a l .

Y i e ld s t r e s s r e f e r s p r i m a r i l y t o t h e i n i t i a t i o n o f p l a s t i c d e f o r m a t io n

above a c e r t a i n l e v e l o f s t r e s s , w h i l e c r i t i c a l s t r e s s i s more g e n e r a l

and i n c l u d e s s h e a r i n g , p l a s t i c and v i s c o u s s t r a i n . One m ajo r

d i f f i c u l t y w i th t h e m easu rem en t o f p l a s t i c and e l a s t i c com ponents in

172

173

r o o t t i s s u e i s t h a t th e t i s s u e i s composed n o t o n ly o f d i f f e r e n t c e l l

ty p e s b u t t h e t i s s u e ( o r o r g a n ) r e p r e s e n t s num erous d e v e lo p m e n ta l

s t a g e s . The i n c r e a s e in p l a s t i c i t y o f t h e r o o t i s r e p r e s e n t e d by a

c o m p o s i te r e s p o n s e o f b o th t h e d i f f e r e n t c e l l t y p e s and d e v e lo p m e n ta l

s t a g e s o f t h e c e l l s . The i n i t i a l ( f i r s t ) i n c r e a s e i n p l a s t i c i t y a t

250 mg may r e p r e s e n t a r e a l change in e x t e n s i b i l i t y o c c u r r i n g in one

c e l l t y p e o f t h e r o o t .

EDTA i n c r e a s e s c e l l w a l l e x t e n s i b i l i t y in V a lo n ia c e l l w a l l

m a t e r i a l i n t h e a b s e n c e o f a p p l i e d s t r e s s . T h is i s n o t t r u e o f F /T

m aize r o o t s . T e p f e r and C le la n d (1 9 7 9 ) h a v e p ro p o se d t h a t CWL o f

a l g a l c e l l s (V a lo n ia and N i t e l l a ) i n v o lv e s c h e m ic a l d i s r u p t i o n o f

d i v a l e n t c r o s s - l i n k s w i t h i n t h e c e l l w a l l w h i l e o a t c o l e o p t i l e CWL i s

enzyme m e d ia te d . S in c e c e l l w a l l l o o s e n i n g o f m a iz e r o o t s i s

a p p a r e n t l y e n z y m a tic w i th a d ep en d en ce upon d i v a l e n t c a t i o n s , t h e

i n a b i l i t y o f EDTA t o i n c r e a s e p l a s t i c i t y i n m aize w i th o u t t e n s i o n

f u r t h e r s u g g e s t s t h a t d i v a l e n t c a t i o n s i n t h e CWL p r o c e s s o f m aize

r o o t s a r e a s s o c i a t e d w i th t h e e n z y m a tic m echanism and do n o t f u n c t i o n

p r i m a r i l y as c r o s s - b r i d g e s i n th e c e l l w a l l .

C a lc iu m r a p i d l y a l t e r s t h e g ro w th r a t e o f i n t a c t m aize r o o t s .

Growth i s r a p i d l y i n h i b i t e d im m e d ia te ly a f t e r a d d i t i o n o f c a lc iu m .

The d u r a t i o n and d e g re e o f i n h i b i t i o n a r e c o n c e n t r a t i o n d e p e n d e n t .

Below 20 mM, c a lc iu m s t i m u l a t e s g ro w th w i th a s t e a d y r a t e a c h ie v e d

30 - 40 m in u te s a f t e r a d d i t i o n . At a l l c o n c e n t r a t i o n s exam ined ,

r e c o v e r y from t h e i n i t i a l i n h i b i t i o n i n v o l v e s r a p i d f l u c t u a t i o n s a b o u t

an i n c r e a s i n g m id p o i n t . The p e r i o d and a m p l i t u d e o f t h e f l u c t u a t i o n s

174

d im i n i s h as a new s t e a d y g ro w th r a t e i s a p p ro a c h e d . The o s c i l l a t o r y

p a t t e r n i s a l s o r e d u c e d by i n c r e a s i n g c o n c e n t r a t i o n s o f c a lc iu m .

The c a lm o d u l in i n h i b i t o r s c h lo r p r o m a z in e and t r i f l u o p e r a z i n e

i n h i b i t r o o t g ro w th by 90 % a t c o n c e n t r a t i o n s o f 50 and 10 pM,

r e s p e c t i v e l y . C o n c e n t r a t i o n s o f 5 and 10 pM CPZ have l i t t l e e f f e c t .

However, t h e s e low c o n c e n t r a t i o n s o f CPZ i n h i b i t g row th r a p i d l y i f th e

r o o t s a r e p r e - t r e a t e d w i th c a lc iu m . The i n h i b i t o r y a c t i o n upon

c a lm o d u l in o f t h e s e p h e n o t h i a z i n e d e r i v a t i v e s i s c a lc iu m d e p e n d e n t , as

i s t h e a c t i o n o f c a lm o d u l in i t s e l f . P r e l i m i n a r y m easu rem en ts show no

i n h i b i t i o n o f r e s p i r a t i o n by t h e s e CaM i n h i b i t o r s . T hese r e s u l t s

s u g g e s t t h a t CPZ and TFP i n h i b i t a c a lm o d u l in - d e p e n d e n t component o f

t h e g ro w th m echanism .

R e le a s e o f p r o t e i n from m aize r o o t s by o s m o t ic shock was found t o

be c o n c o m i ta n t w i th th e l o s s o f a c i d - i n d u c e d g ro w th . T h is o b s e r v a t i o n

s u g g e s t e d a m ethod o f i s o l a t i n g t h e c a lm o d u l in - d e p e n d e n t com plex

in v o lv e d in g ro w th , o r a t l e a s t a m ethod f o r t h e d e m o n s t r a t i o n t h a t

c a lm o d u l in was one o f t h e p r o t e i n s r e l e a s e d by o s m o t ic sh o c k .

A lth o u g h a c a lm o d u l in r e g u l a t e d p r o t e i n has n o t b e e n i d e n t i f i e d ,

c a lm o d u l in was shown t o be p r e s e n t i n t h e o s m o t ic sh o ck medium. A f t e r

t h e i s o l a t i o n o f CaM from t h e OSM, e x t r a c t i o n o f CaM from m aize r o o t

t i s s u e was a l s o u n d e r t a k e n and t h e CaM was found t o r e p r e s e n t a b o u t

0 .3 5 % o f t h e t o t a l p r o t e i n e x t r a c t a b l e from t h e r o o t t i p and

e l o n g a t i o n zone .

I n c o n c l u s i o n , m aize r o o t c e l l w a l l m a t e r i a l p o s s e s s e s s e v e r a l

p h y s i c a l c h a r a c t e r i s t i c s o f b o th a l g a l and c o l e o p t i l e s y s te m s . C e l l

w a l l l o o s e n i n g a p p e a r s t o be e n z y m a tic w i th a d ependence upon c a lc iu m .

C a lc iu m d r a m a t i c a l l y a l t e r s r o o t g ro w th ; s t i m u l a t i n g g row th a t low

c o n c e n t r a t i o n s and i n h i b i t i n g g row th a t h i g h e r c o n c e n t r a t i o n s .

However, even 100 mM c a lc iu m does n o t i n h i b i t r o o t g row th as much as

20 mM c a lc iu m i n h i b i t s s te m o r c o l e o p t i l e g ro w th . P r o t e i n i s r e l e a s e d

from r o o t t i s s u e by o sm o t ic s h o c k . A c id - in d u c e d g row th i s a l s o

i n h i b i t e d by o s m o t ic s h o c k . One o f t h e p r o t e i n s r e l e a s e d by o sm o t ic

shock i s c a lm o d u l in . The d e t e c t i o n o f ATPase a c t i v i t y i n t h e OSM and

i t s s t i m u l a t i o n by low c o n c e n t r a t i o n s o f c a lc iu m s u g g e s t s t h a t one

f u n c t i o n o f t h e OSM c a lm o d u l in may be t h e r e g u l a t i o n o f a p lasm a

membrane a s s o c i a t e d ATPase w hich c o u ld be in v o lv e d i n th e

a c i d i f i c a t i o n o f t h e c e l l w a l l .

The in v o lv e m e n t o f c a lm o d u l in as a com ponent o f t h e m echanism o f

g row th a p p e a r s m ost p r o b a b le and t h e r e s u l t s p r e s e n t e d in t h i s work

s u g g e s t t h a t f u r t h e r r e s e a r c h t o i d e n t i f y t h e p r o t e i n ( s ) r e g u l a t e d by

c a lm o d u l in s h o u ld be u n d e r t a k e n .

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Appendix A: V e r t i c a l S l a b Gel P r o c e d u r e s .

The p r o c e d u r e s i n c l u d e d i n t h i s a p p e n d i x have been u s e d d u r i n g t h e c o u r s e o f t h i s d i s s e r t a t i o n r e s e a r c h . The s t a i n i n g p r o c e d u r e s a r e p r e s e n t e d i n t h e o r d e r o f p e r s o n a l p r e f e r e n c e . I n g e n e r a l , i f you w i s h t o u s e SDS-PAGE as a t e s t f o r sample h o m o g e n e i ty , c o n s i d e r u s i n g s i l v e r s t a i n i n g . Numerous c o n t a m i n a n t s n o t v i s i b l e by s t a n d a r d Coom ass ie s t a i n i n g t e c h n i q u e s a r e r e a d i l y d e t e c t e d by t h e s i l v e r s t a i n i n g methods p r o v i d e d .

P r o c e d u r e f o r P r e p a r a t i o n o f SDS V e r t i c a l S l a b G e l s .

1. P r e p a r e a l l n e c e s s a r y s t o c k s o l u t i o n s l i s t e d u n d e r t h e Laemmli p r o c e d u r e s .

2. C l e a n i n g t h e G l a s s Gel P l a t e s .

a . C l e a n one s e t o f g l a s s g e l p l a t e s f o r ea ch g e l t h a t w i l l be p r e p a r e d (one n o t c h e d and one u n n o t c h e d p l a t e ) .

b . Wash t h e p l a t e s w i t h l i q u i d d i s h soa p and h o t w a t e r , f o l l o w e d by a t h o r o u g h c l e a n i n g w i t h e t h a n o l . Examine t h e p l a t e s c a r e f u l l y t o make c e r t a i n t h a t no g r e a s e r e m a i n s . Rewash t h e p l a t e s i f t h e r e a r e any smudges, smears o r c l o u d i n e s s .

c . R i n s e t h e p l a t e s w i t h 95% e t h a n o l . Use l a r g e Kim-wipe t o w e l s ( o ro t h e r l i n t f r e e , s o f t d i s p o s a b l e t o w e l s ) t o d r y and wash t h ep l a t e s a f t e r r i n s i n g i n e t h a n o l . Do n o t u s e p a p e r t o w e l s w i t h t h e e t h a n o l ( t h e y can be u s e d w i t h soap and w a t e r ) . Wash w i t h e t h a n o l s e v e r a l t im e s and examine t h e p l a t e s a f t e r e a c h wash. C le a n p l a t e s w i l l n o t have any s t r e a k s o r c l o u d i n e s s when h e l d a t an a n g l e u n d e r no rm a l room l i g h t .

d . P l a c e t h e p l a t e s , b e s t f a c e up , on a l a r g e Kim-wipe t o w e l anda l l o w t h e p l a t e s t o a i r d r y . I f t h e r e i s any d o ub t t h a t t h ep l a t e s a r e c o m p l e t e l y f r e e o f g r e a s e , c l e a n t h e p l a t e s w i t h a c e t o n e , f o l l o w e d by e t h a n o l . Wipe t h e p l a t e s t h o r o u g h l y w i t h a l a r g e Kim-wipe.

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e . For a b s o l u t e l y c l e a n p l a t e s , p r e p a r e a c h rom ic a c i d b a t h (25 gp o t a s s i u m d i c h r o m a t e , 750 ml w a t e r , 250 ml c o n c e n t r a t e d s u l f u r i ca c i d ) and a s o l u t i o n o f e t h a n o l s a t u r a t e d w i t h p o t a s s i u m h y d r o x i d e . P l a c e t h e p l a t e s i n a p l a s t i c o r g l a s s t r a y f i l l e d w i t h t h e chromic a c i d . A l low t h e p l a t e s t o s o a k f o r 2 - 4 h o u r s . Remove t h e p l a t e s by r u n n i n g c o l d w a t e r i n t o t h e t r a y . Wash t h e p l a t e s i n d e m i n e r a l i z e d w a t e r and t r a n s f e r them t o a t r a y c o n t a i n i n g p o t a s s i u m h y d r o x i d e s a t u r a t e d e t h a n o l . Soak t h e p l a t e s f o r 1 h o u r . Remove t h e p l a t e s and wash i n d e m i n e r a l i z e d w a t e r . Wash t h e p l a t e s a s i n s t e p s ( a ) - ( b ) .

f . You s h o u l d c l e a n a l l p l a t e s p r i o r t o u s e . S q u i r t a s m a l l amounto f e t h a n o l o n t o e a c h p l a t e and wipe w i t h a c i r c u l a r m o t i o n . Use al a r g e Kim-wipe (do n o t u s e p a p e r t o w e l s ) . A l low t h e p l a t e s t o a i r d r y f o r s e v e r a l m i n u t e s . Avoid u s i n g r e g u l a r p a p e r t o w e l s t o c l e a n g e l p l a t e s ( e x c e p t when u s i n g s o a p ) . G l a s s s c r a t c h e s , and s c r a t c h e d g e l p l a t e s c a n be a b i g h e a d a c h e . Dus t and l i n t can r u i n a g r e a t g e l ; so u s e l i n t f r e e t o w e l s f o r t h e f i n a l c l e a n i n g o f p l a t e s b e f o r e you a s s e m b l e t h e g e l c ham ber .

3 . Assembly o f t h e G l a s s Gel Chamber.

a . Beg in by g r e a s i n g t h e s p a c e r s l i g h t l y w i t h s i l i c o n vacuum g r e a s e .F o r most g e l s , u s e s p a c e r s t h a t a r e 0 . 8 - 1 . 5 mm i n t h i c k n e s s .Make c e r t a i n t h a t you have a comb o f t h e same t h i c k n e s s .

b . S t a r t w i t h t h e s h o r t e r , b o t to m s p a c e r .

c . S p r e a d t h e s i l i c o n g r e a s e e v e n l y . P l a c e t h e s p a c e r f l u s h w i t h t h eb o t t o m edge o f t h e u n n o t c h e d p l a t e .

d . P r o c e e d t o t h e two s i d e s p a c e r s . G r e a s e t h e s e s p a c e r s and mountthem.

e . A f t e r a l l t h r e e s p a c e r s h a v e been p l a c e d on t h e g l a s s p l a t e , c h e c k t h a t t h e s i d e s p a c e r s a r e f l u s h w i t h t h e b o t to m s p a c e r . To p r e v e n t l e a k s a t t h e e dge s o f t h e chamber , add a s m a l l dab o f s i l i c o n g r e a s e a t t h e c o r n e r s (where t h e s i d e s p a c e r s t o u c h t h e b o t t o m s p a c e r ) u s i n g a t o o t h p i c k .

f . P i c k up t h e n o t c h e d p l a t e by i t s e dge s and l a y i t on t o p o f t h eu n n o t c h e d p l a t e . Be s u r e t h a t t h e c l e a n f a c e o f t h e p l a t e f a c e s i n s i d e ( f a c e s t h e c l e a n f a c e o f t h e u n n o t c h e d p l a t e ) .

g . P i c k u p t h e g l a s s g e l chamber and g e n t l y r e s t t h e chamber on i t sb o t t o m e d g e . Make s u r e t h a t t h e b o t to m o f b o t h p l a t e s a r e evenand f l u s h w i t h e a c h o t h e r .

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h . P l a c e t h e chamber on a box o r t e s t t u b e h o l d e r .

i . P l a c e two clamps on e a c h s i d e s p a c e r i n a l t e r n a t i n g s e q u e n c e (one on one s i d e , one on t h e o t h e r ) . Do n o t p l a c e any clamps on t h e n o t c h e d p i e c e o f g l a s s , t h e y b r e a k o f f e a s i l y . The c lamps must r e s t on t h e g l a s s o v e r t h e s p a c e r s and n o t i n s i d e o f t h e s p a c e r s .

j . P l a c e two c la m p s , e q u a l l y s p a c e d , on t h e b o t to m e d g e . Use t h e 2b o t t o m c lamps as f e e t , and s t a n d t h e g l a s s p l a t e g e l chamber up .

k . I f you a r e t o p o u r t h e g e l l a t e r , p l a c e a p i e c e o f p l a s t i c wrapo v e r t h e to p o f t h e chamber t o p r e v e n t d u s t f rom g e t t i n g i n s i d e .

4 . P r e p a r a t i o n o f Running G e l .

You w i l l need s e v e r a l 10 ml p i p e t t e s , 3 s y r i n g e s (2 x 20 ml,1 x 50 m l ) , a s u c t i o n f l a s k and a s m a l l ( 100 - 150 ml) b e a k e r . Youw i l l need a s eco n d s u c t i o n f l a s k and b e a k e r f o r t h e s t a c k i n g g e l (youc a n n o t r e - u s e them w i t h o u t t h o r o u g h l y c l e a n i n g th e m ) .

a . F i l l one o f t h e 20 ml s y r i n g e s w i t h w a t e r .

b . P r e p a r e t h e g e l s o l u t i o n a c c o r d i n g t o t h e Laemmli p r o c e d u r e o fy o u r c h o i c e .

c . A f t e r a s p i r a t i n g t h e g e l s o l u t i o n s , add t h e ammonium p e r s u l f a t e w h i l e s w i r l i n g t h e g e l s o l u t i o n i n t h e s u c t i o n f l a s k .

d. R a p i d l y , b u t s m o o t h l y , p o u r t h e g e l s o l u t i o n i n t o a b e a k e r .

e . F i l l t h e l a r g e s y r i n g e w i t h g e l s o l u t i o n . G e n t l y and e v e n l y f i l lt h e chamber t o w i t h i n 1 i n c h f rom t h e t o p .

f . W i th t h e s y r i n g e c o n t a i n i n g w a t e r , g e n t l y o v e r l a y t h e g e l s o l u t i o nw i t h a t l e a s t 1 /2 i n c h o f w a t e r . You c a n f i l l i t a l l t h e way t ot h e to p i f you w i s h .

g . A l low t h e r u n n i n g g e l t o s e t f o r a t l e a s t one h o u r . S l i g h t l y h i g h e r r e s o l u t i o n i s o b t a i n e d i f you remove t h e w a t e r o v e r l a y a f t e r one hou r and r e p l a c e i t w i t h l o w e r g e l b u f f e r which has been d i l u t e d t o 1 / 4 c o n c e n t r a t i o n . A l low t h i s b u f f e r o v e r l a y t o e q u i l i b r a t e w i t h t h e g e l f o r a t l e a s t one h o u r . The r u n n i n g g e l c a n be p r e p a r e d 24 h r s . p r i o r t o u s e .

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5. P r e p a r a t i o n o f E l e c t r o p h o r e s i s Chamber and S t a c k i n g G e l .

a . B e f o r e you p r e p a r e t h e s t a c k i n g g e l ( u p p e r g e l ) , s e t up t h e g e l e l e c t r o p h o r e s i s c ham ber .

b . Run a bead o f s i l i c o n vacuum g r e a s e a ro u n d t h e u p p e r edge o f t h e cha m be r , fo rm ing a ' U 1.

c . F i l l t h e b o t to m r e s e r v o i r o f t h e e l e c t r o p h o r e s i s chamber w i t h e l e c t r o d e b u f f e r .

d . Remove t h e w a t e r o r b u f f e r o v e r l a y b e f o r e you p r e p a r e t h e s t a c k i n g g e l s o l u t i o n .

e . P r e p a r e t h e u p p e r g e l ( s t a c k i n g g e l ) .

f . P o u r t h e g e l and i n s e r t t h e comb. Avoid t r a p p i n g any b u b b l e s u n d e r t h e comb. T h i s c a n be a c c o m p l i s h e d by i n i t i a l l y i n s e r t i n g t h e comb i n a t an a n g l e . A l low t h e g e l t o s e t f o r a b o u t 20 - 30 m i n u t e s . I f you i n t e n d t o u s e t h e a l t e r n a t e s t a c k i n g g e l f o r m u l a t i o n , a l l o w a b o u t 5 m i n u t e s a f t e r t h e g e l has become o pa que .

g . A f t e r t h e s t a c k i n g g e l ha s p o l y m e r i z e d , work r a p i d l y .

h . Leave t h e comb i n p l a c e . P l a c e t h e g e l chamber on a box o r t e s tt u b e h o l d e r and remove t h e b o t to m c la m p s .

i . Remove t h e b o t t o m s p a c e r . Remove t h e r e m a i n i n g c lam ps from t h e g e l chamber .

j . P l a c e t h e g e l chamber i n t h e e l e c t r o p h o r e s i s chamber and p l a c e onec lamp on e a c h s i d e o f t h e chamber .

k . F i l l t h e u p p e r r e s e r v o i r w i t h e l e c t r o d e b u f f e r .

1. Very c a r e f u l l y , u s i n g a s l i g h t s i d e t o s i d e m o t i o n , remove t h es t a c k i n g g e l comb. The s t a c k i n g g e l i s v e r y f r a g i l e so bec a r e f u l . Be s u r e t h a t t h e u p p e r r e s e r v o i r i s f i l l e d above t h en o t c h e d g e l p l a t e b e f o r e r em ov ing t h e comb.

m. F i l l a s y r i n g e f i t t e d w i t h an 18 gauge n e e d l e w i t h e l e c t r o d e b u f f e r . C a r e f u l l y f l u s h e a c h w e l l o f t h e s t a c k i n g g e l w i t h e l e c t r o d e b u f f e r t o remove any s t a c k i n g g e l t h a t has s t a r t e d f o rm in g in t h e w e l l . R e p e a t t h e p r o c e s s f o r e a c h w e l l . A t t a c h t h e e l e c t r o d e s t o t h e chamber ( p o s i t i v e t o t h e b o t t o m ) .

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6. Pre-electrophoresis, Loading of Samples and Electorphoresis.

P r e - e l e c t r o p h o r e s i s o f t h e g e l w i l l g e n e r a t e b o t h a b u f f e rc o n c e n t r a t i o n and pH g r a d i e n t w i t h i n t h e s t a c k i n g g e l . T h i s w i l lr e s u l t i n s h a r p e r bands moving i n t o t h e r u n n i n g g e l more r a p i d l y .

a. Load each well with 5 - 10 pi of Laemmli buffer.

b . B e g in e l e c t r o p h o r e s i s a t 15 mA.

c . A f t e r t h e b romphenol b l u e f r o n t has r e a c h e d t h e i n t e r f a c e b e tw e ent h e u p p e r ( s t a c k i n g ) and low er ( r u n n i n g ) g e l , t u r n o f f t h e c u r r e n t and load y o u r s a m p le . Fo r 0 . 8 - 1 . 0 mm g e l s , a v o id u s i n g more t h a n 20 p i o f s am p le .

d. B e g in e l e c t r o p h o r e s i s o f your s a m p le s a t 15 - 20 mA.

e . A l lo w t h e g e l t o r u n u n t i l t h e b l u e f r o n t i s w i t h i n a t l e a s t1 - 1 . 5 i n c h e s o f t h e b o t to m b e f o r e t a k i n g down t h e g e l . I f you wan t t o r u n t h e g e l s f a s t e r , p l a c e them i n t h e r e f r i g e r a t o r a f t e r t h e sample has e n t e r e d t h e s t a c k i n g g e l . Then t u r n t h e c u r r e n t up t o a b o u t 30 - 35 mA (50 mA maximum). The g e l s w i l l r u n v e r y h o t . So be c a u t i o u s when t o u c h i n g t h e g l a s s .

7. P r o c e d u r e f o r T a k in g Down t h e G e l .

Once t h e bromphenol b l u e f r o n t i s w i t h i n 1 i n c h o f t h e b o t to m o ft h e g l a s s p l a t e , you s h o u l d p r e p a r e t o f i x and s t a i n t h e g e l . S e v e r a lm ethods f o r f i x i n g and s t a i n i n g t h e g e l s a r e i n c l u d e d . S i l v e r s t a i n i n g i s t h e most s e n s i t i v e method p r e s e n t e d and c a n be up t o1 0 ,0 0 0 t im e s more s e n s i t i v e t h a n Coom ass ie s t a i n i n g .

a . Get t h e g e l f i x i n g / s t a i n i n g t r a y , t h e f i x i n g s o l u t i o n , a l a r g ep a p e r t o w e l , a t h i n f l a t edged s p a t u l a , some p l a s t i c wrap , and amagic m a r k e r .

b . T u rn o f f t h e power and w a i t a few s e c o n d s u n t i l t h e n e e d l e s on t h em e t e r s i n d i c a t i n g v o l t a g e and c u r r e n t h a v e d ro p p ed t o z e r o .

c . Remove t h e power c a b l e s f rom t h e g e l s u p p o r t chamber .

d . G r a s p i n g t h e t r a y f i r m l y a t t h e b a c k o f t h e t o p r e s e r v o i r , pou rt h e e l e c t r o d e b u f f e r o u t o f t h e u p p e r and low er r e s e r v o i r .

e . Remove t h e c lamps from e a c h s i d e o f t h e g l a s s p l a t e . Keep one ha nd i n f r o n t o f t h e g l a s s p l a t e s and p r e s s l i g h t l y on t h e p l a t e s ( j u s t so t h a t t h e y d o n ' t pop l o o s e and r u i n t h e g e l ) . A f t e r b o t h

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c lamps have been removed, g r a s p t h e p l a t e s a t t h e b o t to m i n t h e l ow e r r e s e r v o i r by p l a c i n g y o u r f i n g e r s u n d e r t h e b o t to m edge o f t h e g l a s s p l a t e s . S l i d e t h e p l a t e s up . Do n o t p u l l o u t ! ! L e t t h e p l a t e s s l i d e up , as a p a i r , u n t i l t h e y have c l e a r e d t h e 'U' s h a p e d s i l i c o n g r e a s e s e a l . Be c a r e f u l as you s l i d e t h e p l a t e s up . They w i l l be s t i f f a t f i r s t , b u t c a n s t a r t s l i d i n g v e r y q u i c k l y , so be r e a d y f o r them. A l s o , be c e r t a i n t o a p p l y e q u a l p r e s s u r e t o b o t h p l a t e s w i t h y o u r f i n g e r s .

f . Lay t h e g e l down, u n n o t c h e d p l a t e f a c i n g down, on a l a r g e p a p e r t o w e l ( l a r g e K im -w ip e) .

g . Remove one o f t h e s i d e s p a c e r s by p u l l i n g i t t o t h e o u t s i d e o f t h e p l a t e s .

h . I n s e r t a t h i n , f l a t edged s p a t u l a i n t o t h e lower c o r n e r be tw e en t h e g l a s s p l a t e s (where t h e s p a c e r u s e d t o b e ) .

i . U s in g t h e s p a t u l a as a c r o w b a r , p r y t h e p l a t e s a p a r t . The g e l s h o u l d s t i c k t o one o f t h e two p l a t e s ( i f you a r e l u c k y i t w i l l be t h e bo t to m p l a t e ) . S e p a r a t e t h e p l a t e s by l i f t i n g t h e n o t c h e d , u p p e r p l a t e o v e r ( a s t h o u g h i t were on a h i n g e ) , and o n t o t h e t o w e l .

j . Move t h e f i x i n g t r a y c l o s e t o t h e g e l and f i l l i t w i t h t h e f i x e r ( u s u a l l y 40 % MeOH, 10% AcH).

k . Q u i c k l y , and sm o o th ly f l i p t h e g l a s s p l a t e w i t h t h e g e l o v e r t h e t o p o f t h e t r a y and a l l o w t h e g e l t o " r o l l " o f f t h e p l a t e i n t o t h e f i x e r . I f i t w o n ' t budge , g i v e i t a nudge by p e e l i n g one c o r n e r ( b o t t o m ) o f t h e g e l f r e e f rom t h e g l a s s p l a t e .

1. Your g e l i s now i n t h e f i x e r . F o l l o w t h e f i x i n g / s t a i n i n gp r o c e d u r e o f you r c h o i c e . You can l e a v e t h e g e l i n f i x e r i n d e f i n i t e l y .

8 . S t o r a g e o f G e l s W i t h o u t F i x i n g .

I f you do n o t w ish t o f i x you g e l , e i t h e r b e c a u s e you w ish t o s l i c e a ba nd o u t or you d o n ' t have enough f i x i n g t r a y s ( w h a t e v e r ) , you c a n f r e e z e t h e g e l .

a . P l a c e t h e g e l on a c l e a n , u n n o t c h e d g e l p l a t e .

b . Cover t h e g e l w i t h p l a s t i c wrap and p l a c e i t i n a p l a s t i c bag .Use a - 8 0 C f r e e z e r i f a v a i l a b l e .

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c. The gel can be thawed for use by placing the gel in a fixing tray covered with plastic wrap.

d. P l a c e t h e t r a y i n t h e r e f r i g e r a t o r f o r A - 6 h r s . D e h y d r a t i o n can be m in im iz ed by p l a c i n g 2 o r 3 damp p a p e r to w e l i n t h e g e l t r a y , b e tw e en t h e t h e g l a s s p l a t e and t h e b o t to m o f t h e t r a y . Do n o t a t t e m p t t o remove t h e g e l f rom t h e g l a s s p l a t e .

e . A f t e r t h e g e l has thawed f o r s e v e r a l h o u r s , remove t h e damp t o w e l s and f i l l t h e t r a y w i t h f i x i n g s o l u t i o n . The g e l w i l l l o o s e n from t h e g l a s s p l a t e and t h e p l a t e c a n t h e n be removed.

Appendix B: Preparation of Gel Solutions

A l l o f t h e f o r m u l a t i o n s l i s t e d a r e b a s e d upon t h e o r i g i n a l p r o c e d u r e d e s c r i b e d by Laemmli ( 1 9 7 0 ) . T h e r e a r e two u s a g e s o f ' a c r y l a m i d e ' i n t h i s a p p e n d i x . The c a p i t a l i z e d word r e f e r s t o t h e t o t a l o f b o t h t h e a c r y l a m i d e monomer and b i s - a c r y l a m i d e . The l o w e r - c a s e word r e f e r s t o t h e monomer. P e r c e n t a g e o f a c r y l a m i d e may r e f e r t o e i t h e r t h e p e r c e n t a g e o f t o t a l A c r y la m id e o r t h e a c r y l a m i d e monomer. Most commonly, s t o c k s o l u t i o n s a r e d e s i g n a t e d by t h e p e r c e n t a g e o f t o t a l A c r y l a m i d e . A 30% s t o c k s o l u t i o n c o n t a i n s a t o t a l o f 30% A c r y la m id e ( b o t h m onom ers) . The r a t i o must be g i v e n i f t h e p e r c e n t a g e i n f o r m a t i o n i s t o be o f any v a l u e . Laemmli u s e d a r a t i o o f 2 9 . 2 / 0 . 8 f o r t h e a c r y l a m i d e / b i s - a c r y l a m i d e ( N , N ' - m e t h y l e n e b i s a c r y l a m i d e ) monomers. T h e r e a l s o a p p e a r s t o be c o n s i d e r a b l e c o n f u s i o n c o n c e r n i n g t h e a c t u a l p r e p a r a t i o n o f a s t o c k s o l u t i o n . The p e r c e n t a g e o f A c r y l a m id e i n t h e s t o c k s o l u t i o n s r e f e r s t o t h e combined t o t a l p e r c e n t a g e o f b o t h t h e a c r y l a m i d e and b i s - a c r y l a m i d e monomers as a f r a c t i o n o f t h e t o t a l s o l u t i o n . As any c h e m i s t knows, t h i s mus t be a p e r c e n t a g e o f 100%. Some r e s e a r c h e r s add 100 ml o f w a t e r t o t h e w e i g h t o f t o t a l a c r y l a m i d e . T h i s i s n i a v e and d e m o n s t r a t e s a m i s u n d e r s t a n d i n g o f b o t h L a e m m l i ' s p r o t o c o l and t h e d e f i n i t i o n o f p e r c e n t a g e . I f you a r e p r e p a r i n g a 30% s t o c k s o l u t i o n , 30 g A c r y l a m id e p l u s 100 ml o f w a t e r i s 130 g and g i v e s a p e r c e n t a g e of 23%. T h i s may sound p i c k y , b u t d o n ' t compare you r g e l s t o anyone e l s e ' s i f you w i s h t o u s e such a p r o c e d u r e f o r p r e p a r i n g s t o c k s o l u t i o n s .

The m a jo r c o n t a m i n a n t o f t e n found i n a c r y l a m i d e i s a c r y l i c a c i d . I t i s c a r c i n o g e n i c and n e u r o t o x i c . Keep t h e a c r y l a m i d e , b i s - a c r y l a m i d e , and TEMED ( N , N , N ' , N ' - t e t r a m e t h y l e n e d i a m i n e ) r e f r i g e r a t e d . The a c r y l a m i d e s t o c k s o l u t i o n ( s ) s h o u l d a l s o be r e f r i g e r a t e d . T h e s e s o l u t i o n s a r e u s a b l e f o r a b o u t one month . None o f t h e o t h e r r e a g e n t s need be r e f r i g e r a t e d . Avoid r e f r i g e r a t i o n o f t h e 10% SDS s o l u t i o n . The SDS w i l l p r e c i p i t a t e . P r e p a r e t h e ammonium p e r s u l f a t e f r e s h , b e f o r e u s e . T h i s s o l u t i o n i s u s a b l e f o r 24 h o u r s .

A l l s o l u t i o n s s h o u l d be f i l t e r e d . Whatman GF/D, g l a s s f i l t e r s ( 2 . 7 pm p o r e s i z e ) work v e r y w e l l . Avoid u s i n g p a p e r f i l t e r s i f p o s s i b l e . W ith t h e e x c e p t i o n o f t h e A l t e r n a t e S t a c k i n g g e l f o r m u l a t i o n , HC1 and NaOH a r e u s e d t o a d j u s t t h e pH o f t h e s o l u t i o n s .

194

195

Laemmli Gel Preparation Procedures.

I . L a e m m l i ' s f o r m u l a t i o n , s o l u t i o n s and p r o c e d u r e ............................... 196

I I . M o d i f i e d Laemmli f o r m u l a t i o n f o r :a . G r e a t e r g e l s t r e n g t h and r e d u c e d b a c k d i f f u s i o n . . . , 197b . Faster gelling ...................................................................................... 198c . G r e a t e r g e l s t r e n g t h and f a s t e r g e l l i n g ......................... 199

I I I . M o d i f i e d Laemmli f o r m u l a t i o n i n c l u d i n g c a l c i u m o r EDTA.a . L a e m m l i ' s f o r m u l a t i o n .................................................................... 200b . I n c r e a s e d s t r e n g t h .............................................................................. 201c . More r a p i d g e l l i n g ......................................................................... 202d . I n c r e a s e d s t r e n g t h and more r a p i d g e l l i n g ................. 203

IV. S t a c k i n g g e l f o r m u l a t i o n s .a . L a e m m l i ' s f o r m u l a t i o n and c a l c i u m o r E D T A ...................... 204b . A l t e r n a t e s t a c k i n g g e l f o r m u l a t i o n .................................... 205

V. E l e c t r o d e B u f f e r f o r m u l a t i o n .a . L a e m m l i ' s f o r m u l a t i o n .................................................................... 206b . I n c l u s i o n o f c a l c i u m o r EDTA ................................................... 206

VI. Laemmli sample b u f f e r and sample p r e p a r a t i o n .................................... 207

S t o c k s o l u t i o n s

a . 30% (w/w) A c r y l a m i d e : 2 9 .2 g a c r y l a m i d e , 0 . 8 g Bis a c r y l a m i d e in100 g o f s o l u t i o n .

b . 12.5% (w/w) A c r y l a m id e : 10 g a c r y l a m i d e , 2 . 5 g Bis a c r y l a m i d e in100 g o f s o l u t i o n .

c . 10% (w /v ) SDS (1 g SDS i n 10 ml o f s o l u t i o n ) .d . 10% (w /v ) Ammonium p e r s u l f a t e ( 0 . 0 5 g / 0 . 5 ml s o l u t i o n ) .e . 1 M T r i s - B a s e pH 8 . 8 ( 1 2 .1 g / 100 ml s o l u t i o n ) .f . 1 M T r i s - H C l , pH 6 . 8 ( 1 5 . 8 g / 100 ml s o l u t i o n ) .g . 0 . 4 7 M T r i s - B a s e , pH 6 . 9 ( 5 . 7 g /1 0 0 ml s o l u t i o n ) .h . TEMED ( s t r a i g h t , u n d i l u t e d ) .i . 70 % (w/w) g l y c e r o lj . 100 mM EDTA, pH 8 . 8 ( 3 . 7 2 g / 1 0 0 ml s o l u t i o n ) ,k . 10 mM c a l c i u m c h l o r i d e ( 0 . 1 1 g / 1 0 0 ml s o l u t i o n ) .

196

I - L a e m m l i ' 8 f o r m u l a t i o n

The f o l l o w i n g s o l u t i o n s , when added i n t h e volum es g i v e n w i l l y i e l d t h e same g e l f o r m u l a t i o n as g i v e n by Laemmli ( 1 9 7 0 ) . A l th o u g h Laemmli c a l l s f o r w/v s o l u t i o n s , one may i n t r o d u c e t h e min or m o d i f i c a t i o n o f u s i n g w/w f o r t h e p u r i s t . One o t h e r m o d i f i c a t i o n ha s b e e n made. Laemmli u s e s T r i s - H C l f o r b o t h pH 6 . 8 and 8 . 8 s o l u t i o n s . T r i s - b a s e h a s b e e n s u b s t i t u t e d f o r t h e pH 8 . 8 s o l u t i o n .

For 40 ml o f Running Gel

1. 8 1 0 .6 13 16 20 ml S t o c k A c r y la m id e6% 8% 10% 12% 15% g e l s .

2 . 15 ml S t o c k T r i s - B a s e pH 8 . 8 ( o r T r i s - H C l f o r t h e p u r i s t ) .

3 . 0 . 4 ml o f 10% SDS.

4 . 1 6 .5 14 11 .5 8 . 5 4 . 5 ml w a t e r f o r :6% 8% 10% 12% 15% g e l s .

5 . Mix t h o r o u g h l y by g e n t l e s w i r l i n g .

6. 10 p i o f TEMED.

7. A s p i r a t e s o l u t i o n t o remove d i s s o l v e a i r .

8 . Add 0 . 1 ml o f 10% Ammonium p e r s u l f a t e and s w i r l g e n t l y .

9 . I m m e d i a t e l y pou r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .

197

II a. Glycerol substitution for water.

1.

2 .

3.

4.

5.

6 .

7.

8 .

9.

For 40 ml of Runn ing Gel

86 %

10.68%

1310%

161 2 %

15 ml S t o c k T r i s - B a s e pH 8 . 8

0 . 4 ml o f 10% SDS.

16 .56%

148%

11.510%

8 . 512 %

20 ml S t o c k A c ry la m id e 15% g e l s .

4 . 5 ml o f 70% g l y c e r o l 15% g e l s .

Mix t h o r o u g h l y by g e n t l e s w i r l i n g .

10 p i o f TEMED.

A s p i r a t e s o l u t i o n t o remove d i s s o l v e a i r .

Add 0 . 1 ml o f 10% Ammonium p e r s u l f a t e and s w i r l g e n t l y .

I m m e d i a t e l y pou r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .

The g l y c e r o l c o n c e n t r a t i o n s v a r y f o r t h e g e l c o n c e n t r a t i o n , as f o l l o w s :

6% 8% 10% 12% 15% g e l s29% 25% 20% 15% 8%

The p e r c e n t a g e h a s b e e n r ounded t o t h e n e a r e s t u n i t .

198

I I b. In c r e a s e d amounts o f c a t a l y s t .

For 40 ml o f Runn ing Gel

1. 8 1 0 .6 13 16 20 ml S t o c k A c ry la m ide6% 8% 10% 12% 15% g e l s .

2. 15 ml S t o c k T r i s - B a s e pH 8 . 8

3. 0 . 4 ml o f 10% SDS.

4 . 1 6 .3 1 3 .8 1 1 .3 8 . 3 4 . 3 ml w a t e r f o r :6% 8% 10% 12% 15% g e l s .

5. Mix t h o r o u g h l y by g e n t l e s w i r l i n g .




9. I m m e d i a t e l y pou r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .

199

I I c . G l y c e r o l s u b s t i t u t i o n and i n c r e a s e d c a t a l y s t .


1. 8 1 0 .6 13 16 20 ml S t o c k A cry lam ide6% 8% 10% 12% 15% g e l s .


3. 0 . 4 ml o f 10% SDS.

4. 1 6 .3 1 3 .8 1 1 .3 8 . 3 4 . 3 ml o f 70% g l y c e r o l6% 8% 10% 12% 15% g e l s .

5 . Mix t h o r o u g h l y by g e n t l e s w i r l i n g .




9 . I m m e d i a t e l y p o u r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .

200

I I I a . Laemmli'a fo r m u la t io n w ith c a lc iu m or EDTA.


1. 8 1 0 .6 13 16 20 ml S t o c k A c ry la m id e6% 8% 10% 12% 15% g e l s .


3. 0 . 4 ml o f 10% SDS.

4a . 0 . 4 ml o f 10 mM c a l c i u m c h l o r i d e .** OR **

4 b . 0 . 4 ml o f 100 mM EDTA.

5. 16 .1 1 3 .6 11 .1 8 . 1 4 . 1 ml w a t e r f o r :6% 8% 10% 12% 15% g e l s .

6 . 10 p i o f TEMED.



9. Add 0 . 1 ml o f 10% Ammonium p e r s u l f a t e and s w i r l g e n t l y .

10. I m m e d i a t e l y p o u r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .

201

I I I b. I n c r e a se d s t r e n g t h w ith c a lc iu m or EDTA.


1 .

2 .

3.

4a .

4b .

5.

6 .

7.

8 .

9.

10.

86 %

10.68%

1310%

1612 %


0 . 4 ml o f 10% SDS.

0 . 4 ml o f 10 mM c a l c i u m c h l o r i d e . ** OR **0 . 4 ml o f 100 mM EDTA.

16 .16%

13 .68%

11.110%

8. 112 %

20 ml S t o c k A c ry la m ide 15% g e l s .


10 p i o f TEMED.




I m m e d i a t e l y p o u r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .


6% 8% 10% 12% 15% g e l s28% 24% 19% 14% 7%

The p e r c e n t a g e ha s b e e n r o u n d ed t o t h e n e a r e s t u n i t .

202

I I I c . More r a p i d g e l l i n g , same s t r e n g t h w i t h c a l c i u m o r EDTA.

For 40 ml o f R unn ing Gel

1. 8 1 0 .6 13 166% 8% 10% 12%


3. 0 . 4 ml o f 10% SDS.

4 a . 0 . 4 ml o f 10 mM c a l c i u m c h l o r i d e .** OR **

4b . 0 . 4 ml o f 100 mM EDTA.

5. 1 5 . 9 1 3 .4 1 0 .9 7 . 96 % 8% 10% 12%




9. Add 0 . 3 ml o f 10% Ammonium p e r s u l f a t e and s w i r l g e n t l y .

10. I m m e d i a t e l y p o u r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .

20 nri S t o c k A c ry la m id e 15% g e l s .

3 . 9 ml w a t e r f o r : 15% g e l s .

203

I I I d. In c r e a se d s t r e n g t h and f a s t e r g e l l i n g w ith c a lc iu m or EDTA.

F or 40 ml o f R unn ing Gel

1.

2 .

3.

5.

6 .

7.

8 .

9.

10.

86 %

10.68%

1310 %

161 2 %


0 . 4 ml o f 10% SDS.

4 a . 0 . 4 ml o f 10 mM c a l c i u m c h l o r i d e .** OR **

4b . 0 . 4 ml o f 100 mM EDTA.

1 5 .96%

13 .48%

1 0 .910%

7 . 912 %

20 ml S t o c k A c ry la m ide 15% g e l s .


20 p i o f TEMED.




I m m e d i a t e l y pou r t h e s o l u t i o n i n t o t h e g e l c a s t i n g t r a y .


6% 8% 10% 12% 15% g e l s28% 23% 19% 14% 7%

204

IV a . Laemm li1s s t a c k i n g g e l f o r m u l a t i o n .

1. 2 ml o f S t o c k A c r y l a m id e .

2. 0 . 2 ml o f 10 % SDS.

3. 2 . 5 ml o f T r i s - H C l , pH 6 . 8 .

4. 15 ml w a t e r f o r Laemmli o r 1 4 .8 ml o f w a t e r f o r EDTA/Calciuma d d i t i o n .

5 a . No a d d i t i o n . T h i s i s s t r a i g h t Laemmli . Use 15 ml w a t e r i n above .

** OR **

5b. 0 . 2 ml o r 100 mM EDTA.

** OR **

5 c . 0 . 2 ml o f 10 mM C a lc ium c h l o r i d e .


7. S w i r l g e n t l y and a s p i r a t e .

8 . Add 0 . 1 0 ml o f 10% Ammonium p e r s u l f a t e and s w i r l g e n t l y .

9 . F i l l t h e g e l c a s t t o t h e t o p and g e n t l y i n s e r t t h e comb.

205

IV b. A l t e r n a t e S t a c k i n g Gel f o r m u l a t i o n .

T h i s f o r m u l a t i o n s was a b s t r a c t e d from t h e P l a n t P h y s i o l o g y l a b c o u r s e (B634) a t Ohio S t a t e U n i v e r s i t y . The a d v a n t a g e o f t h i s s t a c k i n g g e l o v e r t h e Laemmli f o r m u l a t i o n i s t h a t i t i s opaque and has l e s s t e n d e n c y t o fo rm w i t h i n t h e w e l l s .

Gel s o l u t i o n s :

T r i s - B a s e (470 mM): 5 . 7 g T r i s - B a s e i n 100 ml o f s o l u t i o n , a d j u s t e dt o pH 6 . 9 w i t h c o n c e n t r a t e d (85.5%) p h o s p h o r i c a c i d .

A c r y l a m id e s o l u t i o n , 12.5% (w/w) : 10 g a c r y l a m i d e , 2 . 5 gB i s - a c r y l a m i d e i n 100 g o f s o l u t i o n .

Gel p r e p a r a t i o n , f o r 20 ml:

5 . 0 ml T r i s - B a s e (470 mM)1 0 . 0 ml s t o c k A c r y la m id e s o l u t i o n (12.5% w/w)

4 . 6 5 ml w a t e r 0 . 2 ml SDS, 10%

40 p i TEMED

A s p i r a t e

0 . 1 ml ammonium p e r s u l f a t e , 10%

206

V a . Laemmli e l e c t r o d e b u f f e r

xlO c o n c e n t r a t e d e s c r i p t i o n x l

3 0 .3 g / 1 (FW 1 2 1 . 1 ) (25 mM) T r i s Base pH 8 . 3 3 .0 3 g / 1144 g / 1 (192 mM) G l y c i n e 1 4 .4 g / 1

10 g / 1 (0.1%) SDS 1 .0 g / 1

V b. I n c l u s i o n o f c a l c i u m o r EDTA.

xlO c o n c e n t r a t e d e s c r i p t i o n x l

3 0 .3 g / 1 (FW 1 2 1 . 1 ) (25 mM) T r i s Base pH 8 . 3 3 .0 3 g / 1144 g / 1 (192 mM) G l y c i n e 1 4 .4 g / 1

10 g / 1 (0 .1%) SDS 1 .0 g / 1100 mM EDTA ** OR **10 mM CaCl„ 10 ml

The e l e c t r o d e b u f f e r pH s h o u l d be a d j u s t e d p r i o r t o u s e , e s p e c i a l l y i f i t i s p r e p a r e d by d i l u t i n g a xlO c o n c e n t r a t e . I f c a l c i u m o r EDTA i s added when d i l u t i n g f rom t h e c o n c e n t r a t e , do so p r i o r t o a d j u s t m e n t o f f i n a l vo lum e .

207

VI. Laemmli sample buffer.

6 2 .5 mM T r i s - H C l , pH 6 . 8 2 . 0 % SDS

1 0 .0 % g l y c e r o l (w /v )5.0% 2-MCE ( v / v )0.001% Bromphenol Blue

For 100 ml of Laemmli sample buffer.

0 .9 8 5 g T r i s - H C l , pH 6 . 82 . 0 g SDS

1 0 . 0 g g l y c e r o l4 . 4 8 ml 2-MCE (@ 1 .1 1 6 8 g / m l )1 . 0 ml 0.1% Bromphenol Blue (BPB)

B r in g t o 100 ml w i t h w a t e r .

Us ing e x i s t i n g s o l u t i o n s . . . f o r 16 ml o f sample b u f f e r .

1 . 0 m l , 1 M T r i s - H C l3 . 2 ml, 10% SDS2 . 3 ml, 70% g l y c e r o l0 . 7 ml, 2-MCE (@ 1 .1 1 6 8 g / m l )0 . 1 6 m l, 0.1% BPB

S t o r e i n a c l e a n p l a s t i c b o t t l e . G l a s s b r e a k s , p l a s t i c d o e s ' n t and t h i s s t u f f s t i n k s .

208

Sample p r e p a r a t i o n .

T h i s b u f f e r i s d e s i g n e d t o be added t o t h e sample i n a 1 :1 r a t i o , and i s o n l y f o r SDS g e l s . The g e l sample mus t t h e n be h e a t e d i n a b o i l i n g w a t e r b a t h f o r 2 - 3 m in . P u t t h e s a m p le s i n a w a t e r b a t h a t room t e m p e r a t u r e and h e a t t h e b a t h w i t h a F i s h e r b u r n e r u n t i l i t b o i l s . An i n e x p e n s i v e aluminum s a u c e p a n works much b e t t e r t h a n a g l a s s b e a k e r . Then t u r n o f f t h e b u r n e r and l e t t h e s am p les s i t f o r2 - 3 m i n u t e s . T h i s p r o c e d u r e g i v e s v e r y good r e s u l t s . Be c e r t a i n t o a l l o w t h e s a m p le s t o c o o l t o room t e m p e r a t u r e b e f o r e l o a d i n g them on t h e g e l .

I f you a r e w ork in g w i t h a c r u d e hom ogena te , i t i s b e s t t o c e n t r i f u g e t h e s a m p le s a t 2 0 ,0 0 0 x g f o r 15 min . T h i s w i l l p e l l e t any n o n - s o l u b i l i z e d m a t e r i a l . Remove sam ple o n l y from t h e t o p o f t h e sample s o l u t i o n . I f m a t e r i a l f rom you r sample p r e c i p i t a t e s i n t h e w e l l o f t h e g e l , uneven and s t r e a k e d band p a t t e r n s w i l l be o b t a i n e d . T h e s e a r e p a r t i c u l a r l y n o t i c e a b l e when t h e g e l i s s i l v e r s t a i n e d .

Appendix C: S t a i n i n g o f p o l y a c r y l a m i d e g e l s .

T h r e e s i l v e r s t a i n i n g p r o c e d u r e s a r e i n c l u d e d and l i s t e d i n o r d e r o f p e r s o n a l p r e f e r e n c e . The m o d i f i e d M e r r i l p r o c e d u r e i s r e l i a b l e , r a p i d and v e r y s e n s i t i v e ( a t l e a s t 10 n g ) . The Sammons t e c h n i q u e i s a l s o q u i t e r e l i a b l e and v e r y s e n s i t i v e , b u t i t r e q u i r e s s e v e r a l more s t e p s t h a n t h e M e r r i l t e c h n i q u e . The Sammons s t a i n i s t o u c h i e r , b u t p r o d u c e s an amazing v a r i e t y o f c o l o r s . However , c o l o r s c a n c o n f u s e t h e i s s u e . A n a l y s i s o f what t h e d i f f e r e n c e s i n c o l o r mean i s l i m i t e d . The O a k le y s t a i n , a l t h o u g h v e r y s e n s i t i v e , has n o t been a s r e l i a b l e or r e p e a t a b l e as t h e M e r r i l o r Sammons s t a i n s . The O ak ley p r o c e d u r e u s e s ammonium h y d r o x i d e and s h o u l d be p e r f o r m e d in a hood o r w e l l v e n t i l a t e d a r e a .

M o d i f i e d M e r r i l S i l v e r S t a i n ........................................................................................ 210

M o d i f i e d Sammons S i l v e r S t a i n ...................................................................................... 213

M o d i f i e d O a k le y s i l v e r s t a i n ........................................................................................ 215

D e s t a i n i n g o f s i l v e r s t a i n e d g e l s ............................................................................ 217

Gel D r y i n g .............................................................................................................................. 218

C oom ass ie s t a i n i n g ................................................................................................................ 219

209

M o d i f i e d M e r r i l s i l v e r s t a i n .

See M e r r i l e t a l . (1981) f o r t h e o r i g i n a l p r o c e d u r e . The f o l l o w i n g i s a m o d i f i e d p r o c e d u r e s i m i l a r t o t h a t p r o v i d e d by Bio-Rad L a b o r a t o r i e s ( b u l l e t i n # 1 0 9 8 ) . T h i s p r o c e d u r e w orks . The o r i g i n a l p r o c e d u r e g i v e s v e r y poor r e s u l t s . The vo lumes shown may be v a r i e d , d e p e n d in g upon t h e s i z e o f you r g e l and g e l t r a y . G e n e r a l l y , t h e g e l s h o u l d be w e l l c o v e r e d by t h e s o l u t i o n .

1. F i x i n 40% MeOH, 10% AcH f o r a t l e a s t 30 min. (May be l e f ti n d e f i n i t e l y a t t h i s s t a g e . Use 1 h r f o r g e l s t h i c k e r t h a n 1mm). T y p i c a l l y , t h i s i s a good s t o p p i n g p o i n t i f t h e g e l i s c o m p l e t e d a t t h e end o f t h e da y . Leave t h e g e l o v e r - n i g h t .

2. F i x i n 400 ml o f 10% EtOH, 5% AcH f o r 15 min. The g e l c a n bel e f t i n t h i s s o l u t i o n a lm o s t i n d e f i n i t e l y .

3. F i x i n 400 ml o f 10% EtOH, 5% AcH f o r 15 min.

I f your g e l c o n t a i n s SDS, r e p e a t s t e p s 2 , 3 a t l e a s to n c e .

4. Soak f o r 5 min. I n 250 - 400 ml o f o x i d i z e r s o l u t i o n ( 3 . 4 mMp o t a s s i u m d i c h r o m a t e , 3 . 2 mN n i t r i c a c i d ) . P r e p a r e by d i l u t i n gx 10 c o n c e n t r a t e 1 /10 w i t h d i s t i l l e d w a t e r . I f t h e o x i d i z e r has b e e n u s e d p r e v i o u s l y , i n c r e a s e t h e t im e t o 10 min. O x i d i z e r can be r e - u s e d a t l e a s t 5 t im e s (up t o 10 i f you a v o id c o n t a m i n a t i o n ) .

5 . Wash f o r 5 min. I n 400 ml o f w a t e r (DDW or DW). Do n o t u s e t a p w a t e r u n d e r any c i r c u m s t a n c e !

6. Wash f o r 5 min. I n 400 ml o f w a t e r (DDW or DW). Do n o t u s e t a pw a t e r u n d e r any c i r c u m s t a n c e !

7. Wash f o r 5 min. I n 400 ml o f w a t e r (DDW o r DW). Do n o t u s e t a pw a t e r u n d e r any c i r c u m s t a n c e !

211

8 . Soak in 250 - 400 ml o f s i l v e r n i t r a t e s o l u t i o n (12 mM) f o r 20 m in . E xpose t h e g e l t o a b r i g h t f l u o r e s c e n t l i g h t . T h i s seems t o d e c r e a s e b ack g ro u n d s t a i n i n g o f th e g e l . P r e p a r e f r e s h b e f o r e u s e by d i l u t i n g x 10 o r x 20 c o n c e n t r a t e 1 /1 0 o r 1 /2 0 r e s p e c t i v e l y w i th d i s t i l l e d w a t e r . The s i l v e r n i t r a t e s o l u t i o n c a n be r e - u s e d a t l e a s t 5 x (up t o 1 0 ) . Be c a r e f u l t o a v o id c o n ta m in a t io n w i th t h e O x i d i z e r o r Image d e v e lo p e r .

9. Wash f o r 1 m in . I n d i s t i l l e d ( d e m i n e r a l i z e d ) w a t e r . No lo n g e r !

10. D eve lop f o r no more th a n 30 s e c . ( I u s u a l l y u se b e tw e en 5 - 2 0 s e c . ) in 250 - 400 ml o f Image D e v e lo p e r ( 0 .2 8 M sod ium c a r b o n a t e , 0 .5 m l / l i t e r 37% f o r m a ld e h y d e ) . The s o l u t i o n w i l l become brown and c lo u d y . Avoid l e t t i n g th e s o l u t i o n s i t to o lo n g . D ur ing t h i s f i r s t t r e a t m e n t , t h e Image D e v e lo p e r i s r e a c t i n g w i th e x c e s s s i l v e r n i t r a t e p r e s e n t on t h e o u t e r s u r f a c e o f t h e g e l . D i s c a r d t h i s s o l u t i o n as soon as i t becomes muddy o r m urky.

11. D evelop f o r 5 min ( I u s u a l l y u s e a b o u t 1 - 2 m in . ) in 250 - 400 ml o f Image D e v e lo p e r . D i s c a r d t h i s s o l u t i o n when s e v e r a l p r o t e i n bands become v i s i b l e .

12. D evelop f o r 5 m in . I n 200 ml o f Image D e v e lo p e r . T h is s t e p may be e x te n d e d o r r e p e a t e d u n t i l t h e g e l i s f u l l y d e v e lo p e d o r as d a r k as you w a n t .

13. S to p d ev e lo p m en t by w a sh in g i n 5% a c e t i c a c id f o r 5 - 1 0 m in . I f you u se l o n g e r t im e s ( s u c h as o v e r n i g h t ) , o r h i g h e r c o n c e n t r a t i o n s o f AcH, t h e g e l w i l l l i g h t e n and t h e b a ckg round may become b r i g h t y e l lo w w i t h c o l o r s a p p e a r in g i n s e v e r a l o f t h e b a n d s .

N o te : i f you d o n ' t wash w i th 5% a c e t i c a c i d , t h e g e l may s w e l l .

14. Wash th e g e l s e v e r a l t im e s i n d i s t i l l e d w a t e r . The g e l w i l l l i g h t e n i f i t i s n o t washed enough .

N ote :

a . Maximum s e n s i t i v i t y i s a c h ie v e d when t h e g e l i s ex p o sed t o r e l a t i v e l y i n t e n s e u n i f o r m l i g h t d u r in g t h e f i r s t 5 m in u te s in s i l v e r n i t r a t e . F l u o r e s c e n t l i g h t gave th e b e s t r e s u l t s .

b . I f t h e g e l i s l e f t in t h e 5% a c e t i c a c id o v e r n i g h t c o l o r s w i l l d e v e lo p in t h e bands and t h e b a c k g ro u n d w i l l become q u i t e y e l lo w . The g e l b a c k g ro u n d w i l l a l s o become y e l lo w - o r a n g e and th e bands w i l l be a l i t t l e l i g h t e r . T h i s c a n be u s e d t o y o u r a d v a n ta g e i f

212

you o v e r - s t a i n yo u r g e l .

c . When p r e p a r i n g t h e s i l v e r n i t r a t e c o n c e n t r a t e , e i t h e r x 10 o rx 2 0 , do n o t p r e p a r e more t h a n 100 ml and s t o r e in a 100 ml f l a s k w rapped w i t h a lum inum f o i l . The o x i d i z e r and s i l v e r n i t r a t e can be r e u s e d a t l e a s t 5 t im e s . J u s t be s u r e n o t t o c o n ta m in a t e t h e s o l u t i o n s .

d . I f t h e g e l i s t o be r e - s t a i n e d ( a s f o r c a lm o d u l in b a n d s ) , u s e t h e d e s t a i n i n g and s to p b a t h s o l u t i o n s . Wash t h e g e l t h o r o u g h ly a f t e r u s i n g t h e s to p b a th ( u s u a l l y 5 - 6 c h a n g es o f w a te r f o r a t l e a s t 24 h r s . ) .

S o l u t i o n s

1) 40% MeOH (EtOH), 10% AcH

2) 10% EtOH, 5%, AcH

3) O x i d i z e r c o n c e n t r a t e (x 1 0 ) : 34mM p o t a s s i u m d ic h r o m a te( 1 . Og/lOOml) , 32mM n i t r i c a c i d ( . 2 8 8 ml @ 70% /100m l).D i l u t e 1 /1 0 f o r u s e . P r e p a r e 500 ml i f you i n t e n d t o r e - u s e t h e s o l u t i o n . P r e p a r e 200 ml f o r one t im e u s a g e .

4 ) S i l v e r n i t r a t e c o n c e n t r a t e (x 1 0 ) : 120 mM ( 2 .0 4 g /1 0 0 m l ) ; ( x 2 0 ) :240 mM ( 4 .0 8 g /1 0 0 m l ) . D i l u t e a s n e e d e d . P r e p a r e 500 ml i f you i n t e n d t o r e - u s e t h e s o l u t i o n . P r e p a r e 200 ml f o r one t im e u s a g e .

5) Image d e v e lo p e r : 2 9 .6 8 g /L (30 g /L i s good enough) sodiumc a r b o n a t e , 0 .5 m l/L @ 37% HCHO. P r e p a r e f r e s h b e f o r e u s e . N e a r ly 1 l i t e r i s need ed f o r e a ch g e l . T h is s o l u t i o n can n o t be r e - u s e d . D o n ' t f o r g e t t o add t h e fo rm a ld e h y d e .

213

M o d if ied Sammons s i l v e r s t a i n .

See Sammons e t a l . ( 1 9 8 1 ) . S e n s i t i v i t y i s a b o u t 5 ng . T h is i s by f a r t h e m ost e x o t i c o f t h e t h r e e p r o c e d u r e s . I t c a n be a t l e a s t as s e n s i t i v e as t h e M e r r i l t e c h n i q u e . However, i t r e q u i r e s more e f f o r t t o p r e p a r e t h e s o l u t i o n s and i s n o t q u i t e a s r e p e a t a b l e ( m o s t ly b e c a u s e o f c o l o r v a r i a t i o n s ) as t h e m o d i f i e d M e r r i l p r o c e d u r e . T h is p r o c e d u r e r e q u i r e s more t im e th a n t h e M e r r i l t e c h n i q u e .

1. F ix in 50% EtOH, 10% AcH f o r > 2 h r s . ( u s u a l l y 24 h r s . ) can be s t o r e d h e r e -----

2 . Wash in 50% EtOH, 10% AcH f o r 2 h r s . can be s t o r e d h e r e -----

3 . Wash in 25% EtOH, 10% AcH f o r 1 h r . t w i c e . can be s t o r e d h e r e -----

4 . Wash in 10% EtOH, 0.5% AcH f o r 1 h r . tw ic e can be s t o r e d h e r e -----

5 . E q u i l i b r a t e g e l in d e g a s s e d , s i l v e r n i t r a t e ( 1 . 9 g / L ) f o r a tl e a s t 2 h r s .

6 . R in s e in d i s t i l l e d d e g a s s e d w a te r 3x f o r 10 -20 s e c .

7. Reduce s i l v e r w i th d e g a s s e d R e d u c e r s o l u t i o n f o r < 10 m in. (10min maximum). P r o t e i n s p o t s b e g in t o a p p e a r a b o u t 5 - 6 mina f t e r a d d i t i o n o f t h e R e d u c e r . The s p o t s a r e i n i t i a l l y y e l lo w -b ro w n on a y e l l o w - o r a n g e b a c k g ro u n d . Remove t h e g e l w i t h i n 10 m in u t e s .

8 . Enhance c o l o r w i t h d e g a s s e d T o n e r s o l u t i o n , l h tw i c e .

9 . Wash in T o n e r + 1 0 - 2 5 ml o f g l a c i a l a c e t i c a c i d . I t hasb e e n o b s e rv e d t h a t t h e g e l s expand w i t h t im e and can grow q u i t e l a r g e . T h i s i s p r o b a b l y due t o t h e r e s i d u a l NaOH.T h i s t r e a t m e n t p r e v e n t s g e l s w e l l i n g .

10. Wash e x t e n s i v e l y i n d i s t i l l e d w a t e r .

11. I f t h e g e l h a s become to o d a r k , i t may be l i g h t e n e d by u s in gP o t a s s i u m D ic h ro m a te d e s t a i n i n g s o l u t i o n .

214

S o l u t i o n s

R e d u c e r : 8 7 .5 mg/L ( 4 3 .7 mg/500 m l) sod ium b o r o h y d r id e7 .5 m l/L ( 3 .7 5 m l/5 0 0 m l) o f 37% HCHO

B rin g t o 1 l i t e r w i t h 0 .7 5 N NaOH.

The fo rm a ld e h y d e s h o u ld be added t o t h e r e d u c e r j u s t p r i o r t o u s e .

T one r : 7 .5 g /L sod ium c a r b o n a t e

215

M od ified Oakley S i l v e r s t a i n .

T h is i s one o f t h e f i r s t r e l a t i v e l y i n e x p e n s i v e s i l v e r s t a i n i n g p r o c e d u r e s . T h i s p r o c e d u r e u s e s c o n c e n t r a t e d ammonium h y d r o x id e and s h o u ld be p e rfo rm e d i n a hood o r ( v e r y ) w e l l v e n t i l a t e d a r e a . The s e n s i t i v i t y i s a b o u t 10 n g . I have e n c o u n te r e d d i f f i c u t l y w i th r e p e a t a b i l i t y . The t e c h n i q u e w i l l work b e a u t i f u l l y and th e n s u d d e n ly s t o p . P r e p a r i n g f r e s h s o l u t i o n s seems t h e m ost o b v io u s a n sw e r , b u t even f r e s h s o l u t i o n s d o n ' t a lw ays c u r e t h e p ro b le m (som etim es th e t e c h n i q u e s t i l l w o n ' t w o rk ) . P r e p a r a t i o n o f t h e ammoniac&l s i l v e r r e a g e n t seems t o be d e p e n d e n t upon t h e o r d e r o f r e a g e n t a d d i t i o n . The o r d e r p r o v id e d h a s been t h e m ost s a t i s f a c t o r y . F o r t h e p u b l i s h e d p r o c e d u r e s e e O ak ley e t a l . ( 1 9 8 0 ) .

1. F ix i n 50% MeOH, 100 ml10% AcH, 20 ml40% w a t e r , 80 ml f o r 30 m in .

N o te : f o r SDS g e l t h i s f i x i n g s t a g e s h o u ld a lw ays be u s e d and s h o u ld be e x te n d e d o v e r n i g h t w i th s e v e r a l ch an g es o f f i x i n g s o l u t i o n .

2. Wash in 5% MeOH, 10 ml7% AcH, 14 ml

8 8 % w a t e r , 176 ml f o r a t l e a s t 10 m in, a t l e a s t t w i c e . Wash a t l e a s t 5x i f SDS i s p r e s e n t .

3 . F i x g e l in 10% g l u t e r a l d e h y d e (400 ml 25% g l d + 600 ml w a te r ) f o r 30 m in.

4 . Soak in a s p i r a t e d am m oniacal s i l v e r s o l u t i o n f o r < 15 m in.

5 . Wash s e v e r a l t im e s in d i s t i l l e d w a t e r ( a t l e a s t 5 x ) .

6 . F i x i n a s p i r a t e d c i t r a t e t o n e r s o l u t i o n .

7. Wash s e v e r a l t im e s in d i s t i l l e d w a t e r .

216

P r e p a r a t i o n o f Ammoniacal S i l v e r s o l u t i o n

P r e p a r e e x a c t l y i n t h e f o l l o w i n g o r d e r :

a . Add 1 .4 ( 2 .8 ) ml ammonium h y d r o x id eb . Add enough w a te r t o a l l o w r a p i d s t i r r i n g ( l e s s th a n 70 m l) c . Add 4 .0 ( 8 . 0 ) ml 19.4% s i l v e r n i t r a t e (20 g + 100 ml d i s t i l l e d w a t e r ) .d . Add 2 1 .0 ( 4 2 .0 ) ml 0.36% NaOH

B r in g t o 100 (200) ml w i th w a t e r .

S o l u t i o n s

C i t r a t e T o n e r :0.005% c i t r a t e (Na c i t r a t e o r c i t r i c a c i d ) . T h is s o l u t i o n can

be p r e p a r e d as lOOx c o n c e n t r a t e ( 0 .5 g /1 0 0 m l) .0.019% HCHO ==> 0 .5 ml 37% HCHO/ l i t e r

O v e r s t a i n e d g e l s can be d e s t a i n e d w i th F a r m e r ' s r e d u c i n g r e a g e n t w hich i s g i v e n be lo w , o r w i th t h e d e s t a i n i n g s o l u t i o n .S o l u t i o n A:

7 .5 g /L p o ta s s iu m f e r r i c y a n i d e S o l u t i o n B:

2 0 0 .0 g /L Sodium T h i o s u l f a t e ( p e n t a h y d r a t e ) .

N o te : Use o n ly i n w e l l v e n t i l a t e d a r e a . S o l u t i o n A r e l e a s e s h y d ro g en c y a n i d e . Use g lo v e s when w o rk in g w i th t h e s e s o l u t i o n s .

217

D e s t a in in g o f s i l v e r s ta in B .

The f o l l o w i n g r e d u c e r and s to p b a t h s o l u t i o n s a l l o w one t o d e s t a i n s i l v e r s t a i n e d p o l y a c r y la m id e g e l s . The r e d u c e r works q u i t e r a p i d l y so w atch your g e l . D e s t a i n i n g t a k e s a b i t o f p r a c t i c e . I f you l e t t h e g e l d e s t a i n u n t i l i t lo o k s j u s t r i g h t , t h e n you have d e s t a i n e d to o much ( t h e l a g t im e f o r t h e s t o p b a t h w i l l g i v e you an e v e n l i g h t e r g e l ) . As a r u l e o f thumb, a lw ays d e s t a i n on t h e d a rk s i d e .

A f t e r u s in g t h e s to p b a t h , wash you r g e l t h o r o u g h l y , w i th s e v e r a l c h a n g es o f w a te r (d o u b le d i s t i l l e d ) .

10 x c o n c e n t r a t e o f D e s t a i n e r ( r e d u c e r )

60 g P o ta s s iu m d ic h ro m a te20 ml S u l f u r i c a c i d ( c o n c e n t r a t e d )

B r in g t o 1 l i t e r w i th w a t e r .

S to p B a th : 30 g /1 sod ium s u l f i t e ( n o t r e - u s a b l e ) .

D i l u t e t h e r e d u c e r and u s e i t c a r e f u l l y and o n ly a f t e r d e v e lo p in g o f t h e g e l i s c o m p le te . Use t h e s t o p b a t h f o r 2 - 5 m in u te s t o s to p d e s t a i n i n g . I f more d e s t a i n i n g i s d e s i r e d , p r e p a r e a f r e s h s t o p b a t h , and p l a c e t h e g e l in t h e r e d u c e r . I f d e s t a i n i n g goes f u r t h e r t h a n you w i s h , t h e g e l can be r e s t a i n e d by any one o f t h e t h r e e s i l v e r s t a i n t e c h n i q u e s p r o v id e d . However, i t i s b e s t t o c o m p l e te l y d e s t a i n t h e g e l and r e s t a i n i t u s in g t h e same s t a i n i n g p r o c e d u r e ( a v o id s w i t c h i n gbe tw e en s t a i n t e c h n i q u e s ) . When r e s t a i n i n g a g e l , s t a r t a t s t e p #3f o r t h e M e r r i l s t a i n , . F o r t h e Sammons s t a i n , s t a r t a t s t e p # 4 . Fort h e O ak ley s t a i n , s t a r t a t s t e p #2.

218

G el d r y in g .

G e ls s h o u ld be so a k ed in 3 - 6% g l y c e r o l (w /v ) f o r l h r . , w i th a t l e a s t two ch an g es o f s o l u t i o n s b e f o r e d r y in g t h e g e l . T h i s w i l l p r e v e n t c r a c k i n g . Use a vacuum d r y e r w i th h e a t . T h is w i l l c u t t h e t im e down t o 1 - 2 h r s . The g l y c e r o l s o l u t i o n s h o u ld i n c l u d e 10 - 25% ( v / v ) e t h a n o l t o s h r i n k t h e g e l back t o i t s o r i g i n a l , p r e f i x i n g s i z e .A s o l u t i o n o f 3% (w /v ) g l y c e r o l , 20 % e t h a n o l u s u a l l y works q u i t e w e l l . I f t h e g e l has b e e n d e s t a i n e d , be c e r t a i n t h a t you have u se d t h e s to p b a t h and washed t h e g e l t h o r o u g h l y .

219

C oom assie B r i l l i a n t B lue R-250 (CBBR-250) S t a i n s

A l l o f t h e s e s t a i n p r o c e d u r e s w ork . S e n s i t i v i t y i s a b o u t 1 - 10 pg o f p r o t e i n ( 0 .1 pg i f you a r e r e a l l y g o o d ) . The p r o c e d u r e s a r e p r o v id e d i n d e s c e n d in g o r d e r o f p e r s o n a l p r e f e r e n c e .

1.

S t a i n f o r 1 - 2 h o u r s w i t h :

100 ml MeOH (o r EtOH), 45.5% ( v / v )100 ml w a t e r , 45.5% ( v / v )

20 ml AcH ( g l a c i a l ) , 9% ( v / v )0 .4 4 g CBBR-250, 0.2% (w /v )

D e s t a i n w i th :

50 ml MeOH (EtOH), 5% ( v / v )850 ml w a t e r , 85% ( v / v )100 ml AcH, 10% ( v / v ) u s e s e v e r a l c h a n g es o v e r a b o u t 24 h r s .

2.

S t a i n f o r 1 - 2 h o u r s w i t h :

0 .4 g CBBR-250, 0.2% (w /v )1 0 0 .0 ml MeOH, 50% ( v / v )

2 0 .0 ml AcH, 10% ( v / v )8 0 .0 ml w a t e r , 40% ( v / v )


2 0 0 .0 ml EtOH, 20% ( v / v )1 0 0 .0 ml AcH, 10% ( v / v )7 0 0 .0 ml w a t e r , 70% ( v / v )

220

F i x f o r 2 h o u rs w i th :

106 ml w a t e r , 53% ( v /v )80 ml MeOH, 40% ( v /v )14 ml AcH, 7% ( v / v )

Then s t a i n w i th :

0 .1 g CBBR-250, 0.05% (w /v ) 106 ml w a t e r , 53% ( v /v )

80 ml MeOH, 40% ( v /v )14 ml AcH, 7% ( v /v )

S t a i n f o r a t l e a s t 1 h o u r w i th :

lOOg T r i c h l o r o a c e t i c A cid (TCA), 50% (w /v ) 100 ml w a t e r , 50% ( v /v )

0 . 2 g CBBR-250, 0.1% (w /v )


70 ml AcH, 7% ( v / v )930 ml w a t e r , 93% ( v / v )

S t a i n f o r 1 - 2 h o u r s w i th :

0 .4 g CBBR-250, 0.2% (w /v )20 ml P e r c h l o r i c a c id ( c o n c e n t r a t e d ) , 3.5% ( v / v )

180 ml w a t e r , 96% ( v /v )

No d e s t a i n i n g r e q u i r e d .

221

6.

S t a i n f o r 1 - 2 h o u rs w i th :

0 .1 g CBBR-250 in 10 ml EtOH 3 0 .0 g TCA, 12% (w /v )

220 ml w a t e r , 88% ( v / v )

D e s t a i n by p l a c i n g g e l s i n d i s t i l l e d w a t e r .

7.f o u r s t a g e s

a . S t a i n 24 h r s . w i t h : 0 .1 g CBBR-2505 0 .0 ml i s o p r o p a n o l2 0 .0 ml AcH

1 3 0 .0 ml w a te r

b . S t a i n 6 - 9 h r s . w i th : 20 ml i s o p r o p a n o l20 ml AcH ( g l a c i a l ) 0 .1 g CBBR-250

c . S t a i n o v e r n i g h t w i th : 20 ml AcH ( g l a c i a l )0 .0 5 g CBBR-250

d. D e s t a i n f o r s e v e r a l h o u r s in 10% AcH.

Appendix D: SDS P o ly a c ry la m id e G rad ien t G e l s .

The p r o c e d u r e s g iv e n f o r t h e p r e p a r a t i o n o f g r a d i e n t SDS-PAGE i n c l u d e b o th l i n e a r and e x p o n e n t i a l g r a d i e n t s . The volume o f g e l s o l u t i o n w i l l depend upon th e s i z e o f t h e g e l cham ber p l a t e s and s p a c e r s t h a t w i l l be u s e d t o p r e p a r e t h e g e l . The vo lum es r e q u i r e d s h o u ld be m ea su red by a s s e m b l in g a g e l cham ber and c o m p le te ly f i l l i n g i t w i th w a t e r . R e p e a t t h i s p r o c e d u r e f o r e v e r y s i z e o f s p a c e r s and g e l p l a t e s t h a t you i n t e n d t o u s e . The p r o t o c o l g iv e n i s s u i t a b l e f o r 13 x 13 cm g l a s s g e l p l a t e s w i t h 0 .7 5 o r 0 .8 0 mm s p a c e r s . For o t h e rs i z e s p a c e r s o r g e l p l a t e s , c a l c u l a t e a new volume u s i n g th ep r o p o r t i o n s o f r e a g e n t s l i s t e d f o r a 10 ml s o l u t i o n vo lum e.

The p r e p a r a t i o n o f g r a d i e n t g e l s r e q u i r e s a s m a l l ( c a . 100 m l) g r a d i e n t m ix in g cham ber. A chamber d e s ig n e d f o r t h e p r e p a r a t i o n o f s u c r o s e g r a d i e n t s i s s u i t a b l e . The u s e o f a p e r i s t a l t i c pump f o r f i l l i n g t h e g e l cham ber i s d e s i r a b l e b u t n o t e s s e n t i a l . I have found t h a t g r a v i t y f e e d s works q u i t e w e l l i f t h e h e i g h t o f t h e g r a d i e n t m aker i s a d j u s t a b l e . T h is can be a c c o m p l i s h e d m ost e a s i l y w i th a l a b - j a c k .

The e s s e n t i a l s u p p l i e s f o r t h e p r e p a r a t i o n o f g r a d i e n t g e l s a r e :

2) s m a l l (25 m l) s u c t i o n f l a s k s1 ) p a i r s u r g i c a l f o r c e p s1 ) 20 ml s y r i n g e1) h a n d f u l o f 5 and 10 ml p i p e t t e s 1 ) 1 - 5 u l p i p e t t o r1) 5 - 50 u l p i p e t t o r1) 50 -2 5 0 u l p i p e t t o r1 ) g r a d i e n t m ix in g chamber ( g r a d i e n t m aker)1 ) l a b - j a c k o r p e r i s t a l t i c pump1 ) m a g n e t ic s t i r p l a t e1 ) s m a l l ( m ic ro ) s t i r b a r , t h e t h i n n e r t h e b e t t e r

I n t r a - m e d i c o r t e f l o n t u b in g t o f i t t h e g r a d i e n t m aker.

222

223

P r e p a r a t io n o f L in e a r G r a d ie n t s .

A ssem ble t h e g l a s s g e l cham ber as p r e v i o u s l y d e s c r i b e d . P r e p a r e t h e two g e l s o l u t i o n s as d e s c r i b e d . A s p i r a t e b o th s o l u t i o n s , b u t do n o t add th e ammonium p e r s u l f a t e . A t t a c h a l e n g t h o f I n t r a - m e d i c t u b in g t o t h e g r a d i e n t g e l m ix e r . Cut th e end t o be m ounted i n t h e g e l chamber a t a s l i g h t a n g l e . Mount t h e f r e e end o f t h e t u b in g in one c o r n e r o f t h e g e l cham ber. Use a s m a l l p i e c e o f t a p e t o h o ld t h e t u b in g in p l a c e .

Add t h e ammonium p e r s u l f a t e t o b o th g e l s o l u t i o n s . W ith th e v a l v e c o n n e c t in g t h e two cham bers o f t h e g r a d i e n t m aker c l o s e d , f i l l t h e r e s e v o i r ( n o t t h e m ix in g ) chamber w i th t h e 6% g e l s o l u t i o n .B r i e f l y open th e v a l v e and a l l o w a s m a l l amount o f t h e 6% s o l u t i o n t o f lo w i n t o t h e m ix in g cham ber. T h is i s t o i n s u r e t h a t no a i r b u b b le s a r e t r a p p e d in t h e c o n n e c t io n be tw een t h e two c h a m b e rs . T urn th e s t i r r e r on and add t h e 20% g e l s o l u t i o n t o t h e m ix in g cham ber. I f you a r e u s in g a p e r i s t a l t i c pump, t u r n i t on . I f you a r e u s in g a g r a v i t y f e e d s y s te m , r a i s e t h e g r a d i e n t maker h ig h enough t o i n s u r e a f lo w r a t e o f be tw een 0 .5 and 1 m l /m in u te . F o r g r a v i t y f e e d , g r a d u a l l y r a i s e t h e g r a d i e n t m ix e r a s t h e g e l cham ber f i l l s .

When t h e g e l s o l u t i o n i s 1 in c h from t h e to p o f t h e g e l cham ber,s t o p t h e f lo w from t h e g r a d i e n t maker e i t h e r by t u r n i n g o f f t h e pump o r c la m p in g th e t u b i n g w i t h a p a i r o f s u r g i c a l f o r c e p s . Remove t h e t u b in g from th e g e l chamber and c a r e f u l l y o v e r l a y t h e g e l s o l u t i o n w i t h w a t e r . A llow t h e g e l t o p o ly m e r iz e f o r a t l e a s t one h o u r . I f you w ish t o l e a v e t h e g e l o v e r n i g h t , r e p l a c e t h e w a te r o v e r l a y w i thg e l b u f f e r d i l u t e d t o 1 /4 c o n c e n t r a t i o n .

P r e p a r a t i o n o f E x p o n e n t i a l G r a d ie n t G e l s .

T h is ty p e o f g e l i s b e s t s u i t e d t o t h e e x a m in a t io n o f h i g h e r m o le c u la r w e ig h t p r o t e i n s (5 0 ,0 0 0 Kd o r l a r g e r ) . The low m o le c u la r w e ig h t ban d s a r e c om pressed e v e n more th a n i n a l i n e a r g r a d i e n t .

P r e p a r e th e g e l chamber and g e l s o l u t i o n s a s p r e v i o u s l y d e s c r i b e d . Add t h e 6% g e l s o l u t i o n t o t h e r e s e v o i r and v e n t t h e c o n n e c t io n be tw een t h e two c h a m b e rs . B e f o re a d d in g t h e 20% g e l s o l u t i o n c l o s e o f f t h e t u b in g c o n n e c t in g t h e g r a d i e n t m aker t o t h e g e l cham ber w i th a p a i r o f f o r c e p s . Add t h e 20% g e l s o l u t i o n t o t h e m ix in g cham ber and s t o p p e r t h e m ix in g cham ber w i t h a c o rk w hich has an 18 gauge ( o r l a r g e r ) n e e d le i n s e r t e d th r o u g h i t . A f t e r t h e m ix ing cham ber i s s t o p p e r e d , remove t h e n e e d l e . The s t o p p e r w i l l m a i n t a i n t h e volume l e v e l w i t h i n t h e m ix in g cham ber. The n e e d le i s n e c e s s a r y t o remove t h e p r e s s u r e b u i l t up d u r in g t h e i n s e r t i o n o f t h e c o r k . Remove th e f o r c e p s and f i l l t h e g e l chamber in t h e same manner as f o r a l i n e a r g r a d i e n t .

224

Gel s o l u t i o n f o r 18 ml t o t a l volume (9 ml e a c h ) .

6% g e l ________________ s o l u t i o n _____________ 20% g e l

1 .8 <======== s t o c k A c ry la m id e ====> 4 .5 ml

3 .4 <======== T r i s - b a s e ===========> 3 .4

90 u l <======== SDS, 10% ============> 90 u l

3 .7 <====== W a t e r / G l y c e r o l , 70% ===> 1 .0

3 u l <======== TEMED ===============> 3 u l

30 u l <===== Ammonium p e r s u l f a t e ====> 30 u l

Gel s o l u t i o n f o r 20 ml t o t a l s o l u t i o n volume (10 ml e a c h ) ,

*6% g e l ________________ s o l u t i o n _____________ 20% g e l

2 .0 <======== S to c k A c ry la m id e ====> 5 .0 ml

3 .7 <======== T r i s - b a s e ===========> 3 .7

0 .1 <======== SDS, 10% ============> 0 .1

4 .1 <====== W a t e r / G l y c e r o l , 70% ===> 4 .1

3 .3 u l <========= TEMED ==============> 3 .3 u l

33 u l <===== Ammonium p e r s u l f a t e ====> 33 u l

*S to c k s o l u t i o n f o r 20% s o l u t i o n i s 40% A c ry la m id e ( 3 8 . 9 / 1 . 1 ) . S to c k s o l u t i o n f o r 6% s o l u t i o n i s 30% A c ry la m id e ( 2 9 . 2 / 0 . 8 ) .

Appendix E: P h o s p h o d ie s te r a s e A ssay fo r C alm odulin .

M easurem ent o f c a lm o d u l in em ployed a m o d i f i c a t i o n o f th e p r o c e d u r e d e s c r i b e d by Sharma and Wang ( 1 9 7 9 ) . The a s s a y r e l i e s upon c o u p le d e n z y m a tic r e a c t i o n s . P h o s p h o d i e s t e r a s e (PDE) c o n v e r t s 3 ' : 5 ’ cAMP i n t o 5 ' AMP and 5 ' n u c l e o t i d a s e c o n v e r t s t h e 5 ' AMP i n t o a d e n o s in e and i n o r g a n i c p h o s p h a te . The p r o c e d u r e f o r d e te r m in in g i n o r g a n i c p h o s p h a te i s a m o d i f i c a t i o n o f t h e Sharma and Wang p r o c e d u r e w hich i s b a s e d upon t h e F i s k e - S u b a r r o w t e c h n i q u e . The c u r r e n t p r o c e d u r e u t i l i z e s 4 mU o f PDE. One u n i t o f c a lm o d u l in has b e e n d e f i n e d as t h e amount o f p r o t e i n n e c e s s a r y t o s t i m u l a t e 0 .0 1 2 u n i t s o f p h o s p h o d i e s t e r a s e by 50 % o f m axim al s t i m u l a t i o n in 1 ml o f a s s a y s o l u t i o n . T h u s , c a l c u l a t i o n o f CaM u n i t a c t i v i t y u s in g t h e r e s u l t s from t h i s a s s a y r e p r e s e n t o n l y 1 /3 u n i t o f t r u e CaM a c t i v i t y ( i e . m u l t i p l y y o u r r e s u l t by 3 ) . T h e re a r e two c o n t r o l s w hich m ust be i n c l u d e d when t e s t i n g f o r c a lm o d u l i n . The b a s a l PDE a c t i v i t y w i th no CaM added m ust be i n c l u d e d in e v e r y a s s a y s e t . T h is i s t h e o n ly i n s u r a n c e t h a t a s s a y s ru n a t d i f f e r e n t t im e s w i l l be c o m p a ra b le . I n o r d e r t o d e m o n s t r a t e t h a t t h e p r o t e i n exam ined i s c a lm o d u l in , one m ust show t h a t t h e s t i m u l a t i o n o f PDE by t h e sam ple i s c a lc iu m d e p e n d e n t . The se co n d c o n t r o l m ust show t h a t PDE a c t i v i t y i s r e t u r n e d t o th e b a s a l l e v e l when E6TA i s s u b s t i t u t e d f o r c a l c iu m in t h e p r e s e n c e o f c a lm o d u l i n . I d e a l l y , a sam p le o f b o v in e CaM w hich i s known t o be p u re s h o u ld be u s e d f o r c a l i b r a t i o n o f t h e s t a n d a r d c u r v e s . A s t a n d a r d c u r v e f o r e v e r y new sam p le o f PDE m ust be g e n e r a t e d . P r e s e n t l y , a l l r e a g e n t s , i n c l u d i n g enzymes a r e from Sigma C hem ica l Co. ( S t . L o u i s , Mo).

225

226

Assay Procedure.

1. Add 0 .1 ml A ssay B u f f e r

2 . 20 p i , 4 .5 mM C aC l^ . F i n a l c o n c e n t r a t i o n i s 0 .1 mM.

3. 20 p i 5 ' N u c l e o t i d a s e (@ 0 .1 2 5 U/10 p i ) .

4 . 5 p i PDE (@ 8 . 0 mU/10 p i , 2 .5 p i @ 16 mU/10 p i ) .

5 . 10 pi of 0 - 0 . 2 ug protein sample (Cm).

T o t a l volume (1 - 5) ==> 0 .1 5 5 ml

6 . Add 0 .7 ml w a t e r .

7 . V o r te x b r i e f l y (15 s e c < ) . May be OMITTED.

8 . I n c u b a t e @ 30 C f o r 1 m in.

9 . Add 50 p i , 1 0 .8 mM 3 ' : 5 * cAMP. T o t a l volume ( 1 - 9 ) ==> 0 .9 0 5 ml(0 .9 0 2 5 ml f o r 2 .5 p i in # 4 ) .

10. V o r te x b r i e f l y (15 s e c ) .

11. I n c u b a t e 30 m in . @ 30 C.

12. Add 100 p i 55% TCA ( t o t a l volume i s 1 m l ) .

13. V o r te x t h o r o u g h l y .

14. Centrifuge 10 min @ maximum speed in Beckman Microfuge (or 10-15 K x g for 15 min. to pellet the precipitate).

15. Remove two a l i q u o t s e a ch o f 0 . 4 ml s u p e r n a t a n t .

S te p 16 - 21 a l lo w s 2 a l i q u o t s o f 0 .7 ml e a c h from e a ch t e s t s a m p le .

16. Add 100 p i o f w a te r t o e a ch a l i q u o t t u b e .

17. Add 100 p i M o lybda te s o l u t i o n .

18. Add 100 p i F i s k e - S u b a rro w r e d u c i n g r e a g e n t .

19. V o r te x t h o r o u g h l y .

227

20. I n c u b a t e 10 min @ 25 C (room T ) .

21. M easure a b s o rb a n c e @ 660 nm u s i n g w a te r as a. b l a n k .

A ssay s o l u t i o n s

C a lc iu m ( 4 .5 mM): 6 6 .2 mg C a C ^ i n 100 ml w a t e r .

EGTA ( 4 . 5 mM): 1 7 1 .2 mg EGTA i n w a te r

TCA (55% w /v ) : 55 g TCA in 100 ml s o l u t i o n (45 ml w a t e r ) .

cAMP ( 1 0 .8 mM): 355 mg 3 * : 5 1 cAMP in 100 ml w a t e r . pH a j d u s t e d t o 7 .0w i t h PDE a s s a y b u f f e r .

A ssay B u f f e r : 4 .3 6 g T r i s - H C l , 2 .4 5 g i m i d a z o l e , 0 .6 5 g MgAc_,pH 7 .5

F-S r e a g e n t : 0 . 5 g / 6 . 3 ml ( 0 .7 9 4 g / 10ml o f w a t e r )

M o ly b d a te : 2 .5 g / 100 ml o f 1 .2 5 N s u l f u r i c a c i d .

==> ml c o n c e n t r a t e d l^SO ^ = (125)/(%H2SO^ x 1 0 /9 8 .0 8 )

F o r 97% t h i s s h o u ld be :

==> 1 2 .6 m l/100 ml s o l u t i o n

228

5 ' N u c l e o t i d a s e i s Sigma # N 5880 (E .C . 3 . 1 . 3 . 5 ) from C r o t a l u s a t r o x i s in a b u f f e r o f 10 mM T r i s b a s e , 0 .5 mM MgAc2 » pH 7 .0 a t 0 .1 2 5 u n i t s / 1 0 p i ) . The n u c l e o t i d a s e i s d i v id e d i n t o a l i q u o t s o f 1 .0 ml and s t o r e d a t -8 0 C.

P h o s p h o d i e s t e r a s e i s Sigma # P 0520 (E .C . 3 . 1 . 4 . 1 7 ) from b o v in e h e a r t in a b u f f e r o f 7 .5 mM T r i s - H C l , 0 .3 7 5 mM c a lc iu m c h l o r i d e ,0 .7 5 mM m agnesium a c e t a t e , 0 .7 5 mM i m i d a z o l e , 25% (w/w) g l y c e r o l , pH 7 .0 a t 8 mU/10 p i

The b u f f e r i s p r e p a r e d by t a k i n g 50 ml o f 20 mM T r i s - H C l , 1 mM m agnesium a c e t a t e , 10 mM c a lc iu m c h l o r i d e , 1 mM im id a z o le and d i l u t i n g t o 1 /2 c o n c e n t r a t i o n . 75 g o f t h i s d i l u t e d b u f f e r i s added t o 25 g o f g l y c e r o l (99%). The pH i s a d j u s t e d w i th 1 M KOH t o 7 .0 . P h o s p h o d i e s t e r a s e i s d e g ra d e d by s u c c e s s i v e f r e e z e / t h a w c y c l e . T h is b u f f e r a p p e a r s t o r e t a r d such d e g r a d a t i o n . The a b i l i t y o f t h i s b u f f e r t o r e t a r d PDE d e g r a d a t i o n a p p e a r s t o be p a r t i a l l y d e p e n d e n t upon t h e i n c l u s i o n o f c a lc iu m . The amount o f c a l c iu m p r e s e n t does n o t s i g n i f i c a n t l y a l t e r t h e c a l c iu m c o n t e n t o f t h e a s s a y s o l u t i o n . A f t e r a d d i t i o n o f PDE to a t o t a l a s s a y volume o f 1 m l, t h e c a lc iu m c o n t e n t c o n t r i b u t e d by th e PDE b u f f e r i s 1 .9 pM.

A ssay B u f f e r i s 360 mM T r i s pH 7 .5 , 360 mM i m i d a z o l e , 45 mM m agnesium a c e t a t e .

Appendix F: Assay fo r ATPase A c t i v i t y .

T h is i s th e p r o c e d u r e o b t a i n e d from Sigma f o r t h e i r recommended ATPase a s s a y . P r o c e d u r e was o b t a i n e d from Sigma on 6 / 8 / 8 2 .

1. B u f f e r c o c k t a i l

FW_________ w e ig h t____________volume t o be added

( 1 2 1 .1 ) 0 . 61g /100 ml( 7 4 .6 ) 2 . 2 4 g / 10 ml( 5 8 .5 ) 1 .4 6 g / 10 ml( 2 0 3 .3 ) 1 .8 0 g /1 0 0 ml

A d ju s t pH t o 7 .4

13 .5 0 ml 50 mM T r i s0 .1 5 ml 3 .0 M KCl1 .5 0 ml 2 .5 M NaCl0 .7 5 ml 90 mM MgCl22 .1 0 ml w a te r •

2. Combine

@10mU/10pl

(FW 7 9 8 .2 ) 0 . 0 6 4 g / l ml

0 .7 2 ml B u f f e r0 .0 5 ml Enzyme ( o r sa m p le )0 .2 0 ml w a te r0 .0 5 ml 80 mM T r i s ATP (Sigm a # A - 0520)

A d ju s te d t o pH 7 .4 . P r e p a r e f r e s h j u s t b e f o r e u s e .

3. I n c u b a t e 30 min a t 37 C.

4 . M easure a b s o rb a n c e a t 660 nm u s in g a s s a y f o r i n o r g a n i c p h o s p h a te o f F i s k e - S ubarrow m ethod ( s e e A ppendix E ) .

229

Appendix G: Radioimmunoassay (RIA) fo r C alm odulin .

T h is p r o c e d u r e u t i l i z e s t h e New E n g la n d N u c le a r (NEN) c a lm o d u l in RIA k i t (#NEK-018) w i t h m o d i f i c a t i o n s t o t h e p r o t o c o l . The sam p les w ere m ea su red u s i n g a Beckman s c i n t i l l a t i o n c o u n t e r ( s e r i e s 7000) ££ggrammed f o r a u to m a t i c quench c o r r e c t i o n and p e r c e n ta g e b i n d in g w i th

I as t h e i s o t o p e .

I . RIA com ponents s u p p l i e d by NEN:

A. C a lm o d u l in A ssay b u f f e r (50 m l) g/lOOml**** 0 .1 2 5 M b o r a t e pH 8 .40 .4 3 8 0 .0 7 5 M NaCl (FW 5 8 .4 4 )0 .2 0 0 .2 % BSA1 ml 1 mM EGTA (FW 3 8 0 .4 )0 .1 0 .1 % sod ium a z i d e s t o r e a t

**** B o r a te b u f f e r i s p r e p a r e d f rom b o r i c a c i d , FW 6 1 .8 3 ,( 0 . 773g /100m l) and sod ium t e t r a b o r a t e , FW 3 8 1 .4 , (4 . '7 7 g /1 0 0 m l) . The sod ium b o r a t e i s u se d t o t i t r a t e t h e b o r i c a c i d t o pH 8 . 4 .

EGTA i s p r e p a r e d as lOOmM ( 0 . 3 8 g /1 0 m l ) .

125B. I - c a l m o d u l i nadd 11 ml CaM a s s a y b u f f e r ( a )

C. C a lm o d u l in - a n t ib o d yadd 11 ml CaM a s s a y b u f f e r ( a )

D. C a lm o d u l in s t a n d a r dadd 2 .0 ml CaM a s s a y b u f f e r ( a ) . C o n c e n t r a t i o n i s

lp g /m l .

E. C a lm o d u l in s e co n d a n t i b o d y d i l u e n t (55 m l)

F. C a lm o d u l in s e co n d a n t i b o d yadd a l l o f ( e )

T o t a l volume o f a s s a y b u f f e r u s e d ==> 24 ml

230

231

I I . D i l u t i o n scheme fo r p r e p a r a t io n o f standard

The s u g g e s t e d volume in t h e d i l u t i o n scheme g iv e n by NEN has been r e d u c e d t o one h a l f .

A = u n d i l u t e d s t a n d a r d AB = A ssay b u f f e rc o n c e n t r a t i o n ( n g / 0 .1 m l)

a . 0 .5 0 ml A + 0 ml AB 100b . 0 .2 5 ml a . + 0 .2 5 ml AB 20c . 0 .2 5 ml b . + 0 .2 5 ml AB 10d . 0 .2 5 ml c . + 0 .2 5 ml AB 5e . 0 .2 5 ml d. + 0 .2 5 ml AB 2 .5f . 0 .2 5 ml e . + 0 .2 5 ml AB 1.25g . 0 .2 5 ml f . + 0 .2 5 ml AB 0 .6 2 5h . 0 .2 5 ml g- + 0 .2 5 ml AB 0 .3 1 3

I I I . A ssay P r o c e d u r e

1. P r e p a r e s t a n d a r d s p r i o r t o u s e (NEN s u g g e s t s im m e d ia te ly b e f o r eu s e , how ever t h i s may n o t be n e c e s s a r y ) .

y

2. P r e p a r e sam ple t u b e s i n a t l e a s t d u p l i c a t e ( p r e f e r a b l y t r i p l i c a t e , u s i n g 1 .5 ml SNAP-CAP v i a l s .

3. F o r s t a n d a r d s i n c l u d e t h e f o l l o w i n g tu b e s w i t h e a ch com ponent as g i v e n :

Tube # B u f f e r S td Sample T r a c e r A n t ib o d y

1 ( t r a c e r ) — — ----- 50 —

2 ( b l a n k ) 100 — ----- 50 —3 ( " 0 " s t d ) 50 — ----- 50 504 ( s t d b . ) — 50 b . ----- 50 505 ( s t d c . ) — 50 c . ----- 50 506 ( s t d d . ) — 50 d . ----- 50 507 ( s t d e . ) — 50 e . ----- 50 508 ( s t d f . ) — 50 f . ----- 50 509 ( s t d g . ) — 50 g . ----- 50 50

10 ( s t d h . ) — 50 h . ----- 50 50

To # 2, add 100 p i o f A ssay B u f f e r ( A . ) .

To # 3 , add 50 p i o f A ssay B u f f e r .

To a l l sam ple t u b e s , add t e s t sa m p le . F i n a l volume m ust be e x a c t l y 50 p i .

232

To #4 th ro u g h 10, add 50 p i o f s t a n d a r d a . - h . ( a . t o # 4 e t c . ) .

To a l l t u b e s , add 50 p i o f T r a c e r ( B . ) .

To t u b e s # 3 on , add 50 p i o f a n t i b o d y .

4 . V o r te x each tu b e

5 . I n c u b a t e a l l t u b e s o v e r n i g h t a t 4 C.

6 . S to p i n c u b a t i o n and v o r t e x a l l t u b e s .

7 . P l a c e i n i c e b a t h .

8 . Add 250 p i o f s e co n d a n t i b o d y t o tu b e # 3 on .

9 . V o r t e x .

10. I n c u b a t e f o r 1 h o u r on i c e .

11. C e n t r i f u g e a t 5 ,0 0 0 x g f o r 15 m in .

12. Add 100 p i o f s u p e r n a t a n t t o e a c h s u p e r n a t a n t sam ple v i a l .

13. D i s c a r d r e m a in in g s u p e r n a t a n t i n t o r a d i o a c t i v e w a s te c o n t a i n e r .

14. S uspend p e l l e t i n s c i n t i l l a t i o n c o c k t a i l (10 m l ) .

15. C ount sa m p le s u s i n g p ro g ra m # 9 w i th second to w e r .

A ppend ix H: P ro g ram L i s t i n g o f D a ta A q u i s i t i o n P rogram , AQUIRE/BAS.Used t o M easure Root G row th.

T h is p ro g ram was w r i t t e n in M i c r o s o f t I n t e r p r e t e r BASIC ( v e r s i o n 5 .0 2 ) on an Im s a i PCS-42 m ic r o c o m p u te r . E x c e p t f o r m o d i f i c a t i o n s o f t h e A/D, k e y b o a rd s c a n , and g r a p i c s d i s p l a y , t h i s p rog ram s h o u ld o p e r a t e on any 8 b i t m ic ro c o m p u te r t h a t u s e d CP/M, v e r s i o n 1 .4 o r l a t e r , as t h e o p e r a t i n g s y s te m .

10 '20 ' AQUIRE.BAS30 ' A/D SAMPLE,PRINT AND DISK STORE OF DATA40 '50 DEF USR0=&HC738:DEF USRl=&HC7D5:0UT 1 3 1 ,060 1 V e r s io n 1 1 .4 0 7 /2 9 /8 1 (C) by Konrad M. Kuzmanoff 1983, a l l

r i g h t s r e s e r v e d70 PRINT CHR$( 2 6 ) ; CHR$( 1 0 ) ; CHR$( 1 0 ) ; CHR$( 1 0 ) ; SPC( 3 0 ) ; "AQUIRE" ; CHR$(10

;CHR$(10)80 PRINT " T h is p ro g ram w i l l c o n v e r t a n a lo g s i g n a l s from two c h a n n e ls

i n t o "90 P R I N T " d ig i t a l i n f o r m a t i o n and o u t p u t t h e i n f o r m a t i o n t o a h a rd c o p y

d e v i c e "100 P R I N T " ( l i n e p r i n t e r ) and t o t h e v i d e o d i s p l a y . The i n f o r m a t i o n i s

s t o r e d on"110 PRINT'M isk a t t h e r a t e o f one r e c o r d f o r e a ch 20 m in u te s o f d a t a .

D a ta "120 P R I N T " a q u i s i t io n o c c u r s a t t h e r a t e o f s i x t im e s / m i n u t e (10 second

i n t e r v a l s "130 PRINT" You may c h o o se t o u t i l i z e b o t h , o r o n ly one A/D

c h a n n e l . I f "140 PRINT"only one c h a n n e l i s s e l e c t e d , i t m ust be c h a n n e l

#1 ." ;C H R $(10 )150 INPUT"ENTER THE NUMBER OF CHANNELS TO BE USED (1 o r 2 ) " ;N155 PRINT CHR$(26);CHR$(10)160 INPUT"Enter t h e c u r r e n t d a t e (MM/DD/YY) ==> ";DT$170 IF MID$(DT$,3 , 1 ) < > " / " OR MID$(DT$,6 , 1 ) < > " / " OR

VAL(M ID$(DT$,1,2))>12 OR VAL(MID$(DT$,4,2))>31 OR VAL(MID$(DT$,7,2))<81 THEN PRINT"ERROR. RE-ENTER":GOTO 160

180 DT$( 1 )=D I$:DT$(2)=DT$: IF CR=999 THEN CR=0:GOTO 2460190 IF N>2 THEN PRINT"ERROR. TOO MANY CHANNELS. TRY AGAIN":GOSUB

1 7 6 0 :RUN200 DEFINT I : Z=32:T=20:A 0(1)=0:A 0(2)=0:G LC=5

233

234

220 DIM S T S ( 2 ,2 ) ,S T ( 2 ,1 0 ) ,S T $ ( 2 ,1 0 ) ,A 3 0 ( 2 , 6 0 ) ,L T $ (1 8 ) ,S T E R (2 ,6 0 )230 VN=100: L=100: VDN=15: A30=0: CNT%( 1 ) = 1 : CNT%( 2 ) = 1 : NUD%=100 240 '250 PRINT CH R$(26); :FOR 1=1 TO N260 '270 PRINT CHR$(26);"SET TRANSDUCERS FOR 0 DIGITAL OUTPUT OR >127

DIGITAL OUTPUT"280 PRINT"HIT RETURN WHEN READY TO BEGIN ADJUSTMENT OF TRANSDUCER

# " ; I ; : IN P U T ;G$290 PRINT I N P ( 2 9 + l ) , :OUT 20 ,1 6300 L=L+1: IF L>50 THEN PRINT CHR$(26); "HIT ' S ' TO STOP":L=0 310 IF IN P(20)=83 THEN 330 320 GOTO 290330 PRINT CHR$(10):PRINT"IS THIS A SATISFACTORY VALUE (TYPE 'Y ' OR

'N ' ) ? " ;340 IN=IN P(2 0 ) : IF IN<>78 AND IN<>89 THEN 340 350 IF IN=78 THEN 280 360 NEXT I370 PRINT CHR$(26);"Do you w ish t o c h eck t h e s e v a lu e s a g a in (Y/N

re tu rn )" :G O S U B 4030 380 IF X$="Y" THEN 240390 PRINT C H R $ (2 6 ) ;"P la c e you r FORMATTED d a t a d i s k e t t e in d r i v e

B : PRINT"Hit any key t o c o n t in u e " :X $ = IN P U T $ (1 ) :PRINT CHR$(26);395 FOR 1=1 TO N400 PRINT "The f o l l o w i n g f i l e s a r e on y o u r d a t a d i s k e t t e :";CH R$(10) 410 F IL E S "B :* .* "420 PRINT CHR$(10)430 '440 PRINT "ENTER THE NAME OF THE FILE FOR THE EXPERIMENT ON TRANSDUCER

# " ;I:PRINT"(UP TO 6 CHARACTERS) "450 INPUT F $ ( l )460 IF L E N (F $ (I ))> 6 THEN PRINT"ERROR, RE-ENTER":GOSUB 1770:GOTO 440 470 IF CR=999 THEN CR=0:GOTO 2460 480 '490 '500 '510 PRINT "ENTER AN EXPERIMENT SUMMARY (UP TO 100

CHARACTERS)";CHR$(10 ):LINE INPUT ESUM$(I)520 IF LEN(ESUM$(I))>100 THEN PRINT"ERROR, ENTRY TOO LONG.

RE-ENTER":GOSUB 1770:GOTO 510 530 IF CR=999 THEN CR=0:GOTO 2460 540 GOSUB 2460 550 TR $(l)= C H R $(l)555 PRINT CHR$(2 6 ) ; "TRANSDUCER # " I ; " f i l e n a m e i s ==> " ; F $ ( l )560 NEXT I570 PRINT CHR$(26);"Do you w ish t o u s e a p r e v i o u s c a l i b r a t i o n (Y/N)

:GOSUB 4030 580 IF X$="N" THEN 610

235

590 GOSUB 5270:GOSUB 5320:PRINT: PR IN T"Prev ious c a l i b r a t i o n d a t e i s ==>";DATE$;CHR$(10):GOSUB 4050:PRINT"Do you w ish t o u s e t h i s c a l i b r a t i o n (Y/N) ";:GOSUB 4 0 3 0 : IF X$>"N" THEN 1070

600 '610 FOR I=N TO 1 STEP -1620 '630 PRINT CHR$(2 6 ) ; "ENTER THE MINIMUM VALUE OF THE READING ON

TRANSDUCER #" ;I :IN P U T YMIN(l)640 PRINT CHR$(26); " I have a minimum v a lu e o f" ;Y M IN (I ) ; " mm.";CHR$(10) 650 INPUT"Is t h i s c o r r e c t (Y/N e n t e r ) " ; G $ : IF G$<"Y" THEN 630 660 PRINT:PRINT"SET TRANSDUCER # " ; I ; " FOR MINIMUM VALUE TO BE

READ.":INPUT"HIT ENTER WHEN READY";G$670 FOR C%=1 TO 1000 680 OUT 20 ,1 6 690 GOSUB 1550 700 A30=A30+A 710 NEXT715 A30=A30/1000:GOSUB 1570720 XMIN(l)=A30 730 A30=0740 PRINT C H R $ (2 6 ) ;S P C (3 0 ) ;"T ra n sd u c e r #" ;I ;C H R $(10):IN P U T "ENTER THE

MAXIMUM VALUE OF THE READING";YMAX(l)750 PRINT C H R $ (2 6 );" I have a maximum v a l u e o f " ;Y M A X ( l ) m m ." ;C H R $ ( 1 0 ) 760 INPUT"Is t h i s c o r r e c t (Y/N e n t e r ) " ; G $ : I F G$<"Y" THEN 740 770 PRINT"SET TRANSDUCER FOR MAXIMUM VALUE TO BE READ.":INPUT " HIT

ENTER WHEN READY";G$780 FOR C%=1 TO 1000 790 OUT 2 0 ,1 6 800 GOSUB 1550 810 A30=A30+A 820 NEXT C%825 A30=A30/1 0 0 0 :GOSUB 1570 830 XMAX(I)=A30 840 A30=0850 M (I)=(Y M A X (I)-Y M IN (I))/(X M A X (I)-X M IN (I)):

B (I )= Y M IN (I ) - (M (I)* M IN (I ) )860 PRINT CHR$(26); "FOR TRANSDUCER # " ; I870 PRINT"THE UPPER LIMIT IS";Y M A X (l): PRINT"THE LOWER LIMIT

IS ";Y M IN (I)880 VR(I)=ABS(XMAX(I)-XMIN(I))890 PRINT"THIS CORRESPONDS TO A VOLTAGE RANGE OF 900 PRINT USING " # # .# # " jV R ( I )910 '920 '930 '940 M VIDY(l)=-99/(XM AX(l)-XM IN(l))950 '960 BVIDY(I)=(I-1)*100-(M VIDY(I)*XM AX(I))+1

236

970 '980 PRINT:PRINT"Hit any key t o continue":GO SUB 4030 990 NEXT I1000 PRINT:PRINT"Do you wi9h t o r e d o t h e c a l i b r a t i o n (Y/N) ";:G0SUB

4 0 3 0 : IF X$>"N" THEN 610 1010 '1020 GOSUB 5270: ' STORE CALIBRATION1030 LSET YMAX1$=MKS$(YMAX(1)):LSET YMAX2$=MKS$(YMAX(2)) :LSET

YMIN1$=MKS $ (YMIN( 1 ) )1040 LSET YMIN2$=MKS$(YMIN(2)):LSET XMAXl$=MKS$(XMAX(l)):LSET

XMAX2$=MKS$(XMAX(2)):LSET XMIN1$=MKS$(XMIN(1)):LSET XMIN2$=MKS$(XMIN(2 ) )

1050 LSET M1$=MKS$(M(1)):LSET M2$=MKS$(M(2 ) ) :LSET B1$=MKS$(B(1 ) ) :LSET B2$=MKS$(B( 2 ) ) :LSET MVIDY1$=MKS$(MVIDY(1)):LSET MVIDY2$=MKS$(MVIDY(2 ) ) :LSET BVIDY1$=MKS$(BVIDY(1 ) ) :LSET BVIDY2$ =MKS$ (BVIDY( 2 ) ) : LSET D$=DT $ ( 1 )

1060 PUT 1 ,1 : CLOSE1070 GOTO 5 0 9 0 : ' SET UP GLITCH CATCHER1080 GOSUB 4 0 9 0 : ’ SET UP GRAPH LETTERS1090 GOTO 30601100 PRINT CHR$( 2 6 ) ;S P C ( 2 0 ) ; "PREPARE TO ENTER DATA AQUISTITION

INITIATION";CHR$(10)1110 '

1120 INPUT"HIT ENTER WHEN READY FOR READING TO COMMENCE";G$1130 PRINT CH R$(26);1140 FOR D%=1 TO 601150 FOR 1=1 TO N1160 OUT 2 4 , 0 : BRATE%=0:NUD%=100 1170 GOSUB 6 0 0 0 : 'GET THE DATA VALUES FROM THE A/D 1180 SD(I)=((S2/NUD%) - (S1*S1 ) / (NUD%*NUD%) ) * (NUD%/(NUD%-1) ) : IF

S D ( l X .0 0 1 THEN S D (I)= 0 1185 S D (l)= S Q R (S D (l) ) :STER (l,D % )=(1-((S1 /N U D % -SD (I)) / (S1/NUD%)) )*100 1190 IF FIRST<999 THEN A 0(l)=X M IN (l)-A 301200 '1210 IF A30>XMAX(I)*.8+1000 THEN BRATE%=999:PRINT SPC(15);"===>

R e a d j u s t t r a n s d u c e r # " ; I ; " <===":VN=VN+1: ' CHECK FOR OVERRANGE1220 IF FIRST<999 THEN 1240 1230 GOSUB 5 4 1 0 : IF GLITCH(I)<1 THEN 1240 1235 IF GLITCH( I ) > 2 THEN 1247 1237 NUD%=20: GOTO 1170 1240 A30(I,D% )=A30+A0(I)1245 ATEMP(I)=A30+A0(l):GOTO 12501247 GLITCH( I)=999:A30(I,D% )=-999:GOSUB 5460 1250 Y (I)=A T EM P(I)*M (I)+B (I)1270 IF PST%(I)>0 THEN BRATE%=999 1280 IF DSK%>0 THEN DSK%=0 1290 '

237

1340 ' GRAPHING1350 IF GLITCH(I)>0 THEN 1405 1360 Y=MVIDY(I)*A30(I,D%)+BVIDY(l)1370 IF Y>I*100 THEN Y=Y-99:GOTO 1370 1380 IF Y < (I-1 )* 1 0 0 + 1 THEN Y=Y+99:GOTO 1370 1390 IF Z>240 THEN U=USRl( 0 ) :U=USR0(0):Z=32 1400 GOSUB 5 2 5 0 : 'DRAW THE POINTS 1405 GOSUB 1 7 6 0 : 1 CALL TIMER CHECK1410 NEXT I1420 1 = 1 :GOSUB 1 8 0 0 : ' PRINTOUT ROUTINE1430 Y 2 ( l ) = Y ( 1 ) : Y 2(2)=Y (2)1440 Z=Z+11465 GOSUB 2 6 0 0 : ' CALL SERVICE INTERRUPT1470 GLITCH( 1 )= 0 : GLITCH( 2 ) = 0 : T=T+20: NEXT D%1480 ' DISK STORAGE ROUTINE CALL AND CHECK1490 '1500 FOR 1=1 TO N: GOSUB 2150:GOSUB 2200:NEXT 1510 '1520 '1540 GOTO 1 1 4 0 : ' RETURN TO DATA AQUISITION1550 ' DATA AQUISITION1560 A = IN P ( l+ 2 9 ) : RETURN1570 IF A30>127 THEN A30=A30*.0 2 - 5 .1 2 ELSE A30=.02*A30 1580 RETURN1590 '1600 '1760 ' DELAY ROUTINE1770 IF BRATE%>0 THEN OUT 2 0 ,1 61780 IF IN P(24)>128 THEN 1 7 7 0 : ' CHECK TIMER1790 RETURN1800 1 VIDEO ROUTINE1810 11820 FOR L=1 TO N1830 IF S T S (L ,0 )> 0 AND S T S (L ,1 )> 0 THEN 1 8 5 0 : 'DESC STAT AND TIME

SPECIFIED 1840 GOTO 19501850 IF T>=(ST(L,CNT% (L))-300) THEN 18701860 GOTO 19501870 IF PST%(L)>0 THEN 18901880 PST%(L)=999: VDN=VDN-3:VN=1001890 IF T<(ST(L,CNT%(L)))+20 THEN 19301900 CNT%(L)=CNT%(L)+1: IF CNT%(L)>STS(L,1) THEN S T S (L ,0 )= 0 1910 PST%(L)=0:VDN=15:VN=100 1920 '1930 NEXT L1940 '1950 IF VN<VDN+1 THEN 2020 1960 PRINT CHR$(26);:VN=0

238

1970 FOR L-l TO N1980 IF PST%(L)>0 THEN P R IN T "T ransducer # " ; L ; S P C ( 5 ) ; " S p e c i f i e d t im e i s

===> ";ST(L,CNT%(L)):PRINT ST$(L,CNT%(L))1990 '2000 NEXT2010 PRINT SPC(3) ; " F i l e name i s ==> " ; F $ ( 1 ) ; S P C ( 2 0 ) ; " F i l e name i s ==>

" ;F $ (2 ) :P R IN T SPC(1 5 ) ; ; "TRANSDUCER # 1 " ;S P C (3 0 ) ; "TRANSDUCER #2";C H R$(10)

2013 PRINT "Time ( s e c ) " ; S P C ( 5 ) ; " L e n g th " ;S P C ( 5 ) ; " r a t e (m m /hr) " ; SPC( 1 5 ) ; " L e n g th " ; SPC( 5 ) ; " r a t e (m m /hr)"

2015 PRINT STRING$(79,45)2020 VN=VN+1: PRINT USING " # # # # # " ;T ;2030 PRINT SPC (IO );2040 PRINT USING " # # .# # # " ;Y (1 ) ;2050 IF FIRST=0 THEN PRINT STRING$( 2 2 , 3 2 ) ; :GOTO 2075 2060 PRINT SPC( 6 ) ;2070 PRINT USING " # # # . » " ; ( Y ( l ) - Y 2 ( l ) ) * 1 8 0 ;2073 IF GLITCH(I)>0 THEN PRINT"<== G l i t c h " ; ELSE PRINT SPC (IO );2075 IF N<2 THEN PRINT:GOTO 21402078 IF GLITCH( 2)>0 THEN G LITC H (2)=0:PRIN T"G litch ==>";:GOTO 2100 2080 PRINT SPC( 1 0 ) ;2090 '2100 PRINT USING Y ( 2 ) ;2110 IF FIRST=0 THEN PRINT STRING$( 8 , 3 2 ) :GOTO 2140 2120 PRINT S P C (7 ) ;2130 PRINT USING " # # # . # # " ; ( Y ( 2 ) -Y 2 (2 ) )* 1 8 0 2140 FIRST=999: RETURN2150 ' DISK STORAGE ROUTINE2160 '2170 OPEN " R " , 1 , " B : " + F $ ( l ) + " .E X P " ,2 4 0 :OPEN " R " ,2 , "B : " + F $ ( l ) + " .S T E " ,240 2180 FIELD 1 ,2 4 0 AS D$: FIELD 2 ,2 4 0 AS H$2190 RETURN2200 FOR D%=0 TO 592210 FIELD 1 , D%*4 AS DU$,4 AS D$: FIELD 2,D%*4 AS HU$,4 AS H$2220 LSET D$=MKS$(A30(I,D%+1)):LSET H$=MKS$(STER(I,D%+1) )2230 NEXT2240 PUT 1 ,L R (l) :P U T 2 ,L R ( I )2250 CLOSE2260 L R (I)= L R (I)+ 12270 '2280 GOSUB 2 1 5 0 :GET 1 , 1 : FIELD 1 ,2 AS LR$,238 AS DU$:LSET

LR$=M KI$(LR(I)-1)2290 PUT 1 ,1 : CLOSE2300 DSK%=9992310 RETURN2320 ' DISK DATA FILE SET UP2330 'THE FOLLOWING INFORMATION IS STORED FOR EACH FILE2340 'FILE NAME [ F $ ( l ) ] ,F I L E NAME END [ * ] , DATE [ D T $ ( l ) ,8 BYTES],

239

2350 'YMIN,YMAX,XMIN,XMAX,VOLTAGE RANGE [ V R ( I ) ] , AND EXP. SUMMARY [ESUM$(I)]

2360 GOSUB 21502370 FIELD 1 , 2 AS LR$,6 AS F$ , 8 AS D$, 4 AS YMIN$, 4 AS YMAX$, 4 AS

XMIN$, 4 AS XMAX$, 4 AS VR$, 1 AS T $ ,2 AS DR$,4 AS M$,4 AS B$,193 AS ESUM$

2380 LSET F $ = F $ ( I ) :LSET D$=DT$(I) :LSET YMIN$=MKS$(YMIN(l)):LSET YMAX$=MKS$ (YMAX( I ) ) :LSET XMIN$=MKS$ (XMIN(I)):LSET XMAX$=MKS$(XMAX(I)):LSET VR$=MKS$(VR(l)):LSET ESUM$=ESUM$(I) :LSET T$=TR$(I):LSET LR$=MKI$(0)

2390 LSET DR$=MKI$(STS(I,1 ) )2400 LSET M$=MKS$(M(I)):LSET B$=MKS$(B(l))2410 12420 PUT 1 ,1 2430 CLOSE 2440 L R (I)= 22450 RETURN2460 PRINT C H R $ (2 6 );" I have t h e f o l l o w i n g in fo rm a t io n " ;C H R $ (1 0 ) :PRINT 2470 P R IN T " (F ) i le name ===> " ;F $ ( I ) ;C H R $ (1 0 )2480 P R IN T " (C )u r re n t d a t e ====> " ;D T $(I) ;C H R $(10 )2490 P R IN T "(E )x p e r im en t summary ===>";ESUM$(I);CHR$(10):PRINT 2500 PR IN T"Enter t h e l e t t e r [F ,C ,E ,A ] c o r r e s p o n d in g t o t h e i te m t o be

c h a n g e d , "2510 PRINT"or e n t e r ' N ' , f o r no c h a n g e s . "2520 GOSUB 4030 2530 '2540 CR=9992550 IF X$="F" THEN GOTO 4402560 IF X$="C" THEN PRINT CHR$(26); :GOTO 1552570 IF X$="E" THEN PRINT CH R$(26); :GOTO 5102580 IF X$="N" THEN CR=0:RETURN2590 PRINT CHR$(2 6 ) ; "ERROR. TRY AGAIN":GOSUB 1760:GOTO 2460 2600 ' SERVICE INTERRUPT ROUTINE2610 '2620 IF IN P(20)= 73 THEN 2640 2630 RETURN2640 VN=100:PRINT CHR$(26);2650 PRIN T"Enter t h e n a t u r e o f t h e i n t e r r u p t : ";CHR$(10)2660 '2670 P R IN T "(A )d ju s t t r a n s d u c e r t o ' z e r o ' p o s i t i o n on s t r i p c h a r t

r e c o r d e r " ;C H R $ ( 10)2675 PR IN T"(C)hange t h e g l i t c h c a t c h e r l e v e l o f g l i t c h

c a tc h in g " ;C H R $ (10)2680 P R I N T " ( R ) e s ta r t e x p e r im e n t" ;C H R $ (10)2690 P R IN T "(T )e rm in a te e x p e r im e n t" ;C H R $ (10)2695 PRINT"(N)o c h a n g e s . R e tu r n t o D ata A q u is i t io n " ;C H R $ (1 0 )2700 PR IN T"Enter t h e l e t t e r [A ,C ,R ,T ] c o r r e s p o n d in g t o yo u r r e q u e s t or

'N ' f o r re s u m e "

240

2710 X$=INPUT$(1)2720 IF X$="N" THEN PRINT CHR$(26); :VN=100:GOSUB 1800:RETURN2730 IF X$="R" THEN GOTO 29702740 IF X$="T" THEN 28602750 IF X$="A" THEN 27702755 IF X$="C" THEN 70002760 GOTO 26402770 PRINT CHR$(26);SPC(10)"W hich t r a n s d u c e r i s t o be a d j u s t e d (1 o r 2

r e t u r n ) " ; : I N P U T I : IF I>N OR I<1 THEN 2770 2780 AH=A302790 PRINT:PR IN T"Press any key t o c o n t i n u e , AFTER you h a v e made your

a d ju s t m e n t "2800 GOSUB 4030 2810 '2820 GOSUB 6 0 0 0 : 'GET THE DATA VALUES2830 A 0(I)= -A B S(A 30-A TEM P(I)) :IF GLITCH(I)>0 THEN GLITCH(l)=0 2840 GOSUB 1 8 0 0 :RETURN2850 RETURN2860 PRINT CHR$(26);SPC(20)"You have r e q u e s t e d t e r m i n a t i o n o f d a t a

a q u i s i t i o n " ;C H R $ ( 1 0 )2870 INPUT"IS THIS CORRECT (Y/N ENTER)";G$:IF G$<"Y" THEN 2640 2880 PRINT CHR$( 2 6 ) ; "The d a t a f i l e s h a v e been saved u n d e r th e f i l e

n a m e s : " ;CHR$(10)2890 PRINT CHR$(1 0 ) : PRINT"TRANSDUCER #1 ===> " ;F $ (1 ) ;C H R $ (1 0 )2900 PRINT"TRANSDUCER #2 ===> " ;F $ (2 ) ;C H R $ (1 0 )2910 CLOSE:DTEMP%=D%2920 FOR 1=1 TO N :IF DTEMP%<60 THEN

A30(I,DTEMP%+1)=999: STER(I,DTEMP%+1)=999 2930 GOSUB 2150:GOSUB 2200:NEXT2940 PRINT SPC( 2 0 ) ; "THE PROGRAM WILL NOW TERMINATE":PRINT"PROGRAMMED

TERMINATED"2950 INPUT"Do you w ish t o ru n t h e r e t r i e v a l p ro g ram , 'READ' (Y/N

ENTER) ";G$2960 IF G$="N" THEN END ELSE PRINT"Which r e t r i e v a l p ro g ra m do you w ish

t o ru n :" ;C H R $ (1 0 )2961 PRINT" { 1 ) READ2 0 .BAS { f o r 20 second t im e in te r v a l s > " ;C H R $ ( 1 0 )2962 PRINT"{2> READ1 0 .BAS { f o r 10 second t im e in te r v a l s > " ;C H R $ ( 1 0 )2963 INPUT " E n te r y o u r c h o ic e by number (1 o r 2)";CH%2964 IF CH%=1 THEN RUN"READ20. BAS" ELSE RUN"READ10.BAS"2965 END2970 ' PROGRAM RESTART2980 PRINT CHR$(26); "U ser has r e q u e s t e d a p rogram r e s t a r t , i s t h i s

c o r r e c t (Y/N ENTER)"2990 INPUT G$3000 IF G$<"Y" THEN 26403010 PRINT CHR$(26);"Do you w ish t o s a v e th e d a t a f i l e s c r e a t e d d u r in g

d a t a a q u i s i t i o n (Y/N ENTER) ";:INPUT G$

241

3020 IF G$<"Y" THEN PRINT:PRINT"Are you c e r t a i n you w ish t o d e l e t e t h e s e f i l e s

3030 INPUT G$: IF G$>"N" THEN KILL " B :”+ F $ ( l) :K IL L " B : " + F $ ( 2 ) : PRINT"DATA FILES DELETED";CHR$(10)

3040 PRINT CHR$( 2 6 ) ; "P rogram w i l l now re s ta r t" :G O S U B 1760:RUN 3050 '3060 ' DESCRIPTIVE FILE SET UP3070 PRINT CHR$(2 6 ) ; SPC( 2 0 ) ; "DESCRIPTIVE STATEMENT ENTRY";CHR$(10)3080 PRINT"You may e n t e r up t o 10 s t a t e m e n t s d e s c r i b i n g e x p e r i m e n t a l

p r o c e d u r e "3090 PR IN T"for e a ch t r a n s d u c e r . Each s t a t e m e n t m ust be w r i t t e n now,

b e f o r e d a t a "3100 P R I N T " a q u i s i t io n b e g i n s . Each s t a t e m e n t may c o n t a i n any

c o m b in a t io n o f up to "3110 PRINT"80 a l p h a b e t i c , n u m er ic o r p u n c t u a t i o n c h a r a c t e r s . "3120 PRINT" The p u rp o se o f t h e s e s t a t e m e n t s i s t o a l l o w you t o

i n s e r t i n f o r m a t i o n "3130 P R IN T "co n ce rn in g e x p e r i m e n t a l p r o c e d u r e d i r e c t l y i n t o t h e

c o m p u te r r e c o r d e d "3140 PR IN T "da ta . T h is i s i n t e n d e d t o a l l o w you t o more e a s i l y

c o r r e l a t e changes in "3150 PRIN T"the e x p e r i m e n t a l d a t a w i th y o u r e x p e r i m e n t a l p r o c e d u r e

d u r in g r e t r i e v a l "3160 PRINT"of t h e com pu te r g e n e r a t e d d a t a . As an exam ple o f a

d e s c r i p t i v e s t a t e m e n t , "3170 PRINT "you m ig h t i n t e n d t o add 0 .0 8 ml o f 0 .0 1 mM IAA t o t h e t e s t

s o l u t i o n o f "3180 P R IN T " t r a n s d u c e r # 1 , 120 m in . a f t e r t h e e x p e r im e n t was s t a r t e d .

T h i s "3190 P R IN T " in fo rm a t io n c o u ld be e n t e r e d as a d e s c r i p t i v e s t a t e m e n t as

f o l l o w s :" ;CH R$(10)

3200 PRINT"'0 .0 8 ML OF 0 .0 1 MM IAA AT T=120 TO # 1. []= 1 X10E-9 M ' ";CHR$(10)

3210 '3220 PRINT"(Note t h a t an 'E ' m ust be u s e d t o i n d i c a t e a power o f

10)";C H R $(10)3230 PRINT"HIT ANY KEY TO CONTINUE"; : GOSUB 4 0 3 0 :PRINT CHR$(26);3240 PRINT" I f you w is h , you may a l s o s p e c i f y when th e i n f o r m a t i o n

s t a t e m e n t i s "3250 PRINT"to be e n t e r e d i n t o t h e co m p u te r g e n e r a t e d d a t a f i l e . The

t im e you s p e c i f y "3260 PRINT"must be from t h e b e g in n in g o f t h e e x p e r i m e n t . "3270 PRINT" I n t h e above e x a m p le , you would s p e c i f y t= 7 2 0 0 . The

t im e m ust b e "3280 PR IN T"given in s e c o n d s . "3290 PRINT" I f you s p e c i f y a t im e , th e n th e co m p u te r w i l l a l e r t

you , by b e e p i n g , "

242

3300 P R IN T "tha t an i n f o r m a t i o n s t a t e m e n t w i l l be s t o r e d . The s t a t e m e n t to b e "

3310 PRINT"saved w i l l be d i s p l a y e d . The b e e p in g and d i s p l a y o f t h e s t a t e m e n t w i l l "

3320 PR IN T"begin 5 m in . b e f o r e t h e t im e you have s p e c i f i e d . I n t h e above e x a m p le , i f "

3330 PRIN T"the s t a t e m e n t i s t o be s t o r e d a t t= 7200 s e c . , t h e com pute r would b e g in "

3340 P R I N T " s ig n a l l in g you a t t=6900 s e c . (115 m in . ) a f t e r th e b e g in n i n g o f t h e "

3350 P R IN T "e x p e r im e n t . " ;CHR$(1 0 )3360 PRINT"NOTE: I n a l l c a s e s , d e s c r i p t i v e s t a t e m e n t s a r e a c t u a l l y

s t o r e d a t t h e "3370 P R IN T "beg inn ing o f t h e co m p u te r g e n e r a t e d d a t a f i l e " ;C H R $ (1 0 )3380 '3390 GOSUB 4 5 0 0 :GR=999:' GRAPHIC LETTERS3400 '3410 PRINT"HIT ANY KEY TO CONTINUE"GOSUB 4030:PRINT CHR$(26);3420 * SET UP STATEMENT RECORDS3430 FOR 1=1 TO N3440 PRINT CHR$( 2 6 ) ; SPC( 2 0 ) ; "ENTRY OF DESCRIPTIVE

STATEMENTS";CHR$( 1 0 ) ;CHR$(1 0 )3450 PRINT"For t r a n s d u c e r =====> " ;I ;C H R $ (1 0 )3460 PRINT" Do you w ish t o e n t e r any i n f o r m a t i o n s t a t e m e n t s "3470 PRINT"(Y/N) ? " ;3480 GOSUB 4030:PRINT X $:IF X$<"Y" THEN 3830 3490 S T S ( I ,0 )= 9 9 93500 PRINT:PRINT"Do you w ish t o s p e c i f y t h e t im e s o f s t o r a g e o f t h e s e

s t a t e m e n t s (Y/N) ?"3510 GOSUB 4 0 3 0 :PRINT X$: IF X$>"N" THEN S T S (I ,2 )= 9 9 9 3520 '3530 '3540 FOR J=1 TO 103550 PRINT C H R $(26);"For t r a n s d u c e r # " ; I ; " what i s i n f o r m a t i o n

s t a t e m e n t # " ; J 3560 P R IN T "(h i t r e t u r n f o r no e n try ) " ;C H R $ (1 0 )3570 LINE INPUT S T $ ( l , j )3580 IF L E N (S T $ (I ,J ) )> 7 9 THEN PRINT CHR$( 1 0 ) : PRINT"ERROR. ENTRY MUST

BE LESS THAN 80 CHARACTERS IN LENGTH. TRY AGAIN.":GOSUB 1770 :GOTO 3550

3590 PRINT:PRINT C H R $(10);"For i n f o r m a t i o n s t a t e m e n t I have th ef o l l o w i n g :";CH R$(10)

3600 PRINT S T $ ( l , J ) ;CHR$(10)3610 PRINT"Is t h i s c o r r e c t (Y/N) ";:GOSUB 4030:PRINT X $ :IF X$<"Y" THEN

35503620 IF S T $ ( I , J )= "" THEN 3830 3630 S T S ( I , 1 )= S T S (I ,1 )+ 1 3640 IF S T S ( I ,2 )> 0 THEN 3670

243

3650 GOTO 3820 3660 13670 PRINT CHR$( 2 6 ) ; "SPECIFIED TIME MUST BE IN SECONDS AND A MULTIPLE

OF 20";CHR$(10)3680 PRINT"SPECIFIED TIME MUST ALSO BE GREATER THAN T=300"3690 PR IN T"If you i n t e n d t o e n t e r more th a n one d e s c r i p t i v e s t a t e m e n t ,

t h e n "3700 PRINT"the s p e c i f i e d t im e s m ust be a t l e a s t 300 se c o n d s

a p a r t . " ;C H R $ ( 1 0 )3710 IF J>1 THEN PRINT"Last t im e e n t r y was ==> " ; S T ( I , J - l ) ; " s e c o n d s . " 3720 INPUT"At what t im e ( i n s e c . ) s h o u ld t h e s t a t e m e n t be

s t o r e d " ; S T ( l , J )3730 IF S T ( I , J ) < 3 0 0 OR ( S T ( I , J ) / 2 0 ) < > F I X ( S T ( l , J ) / 2 0 ) THEN 3760 3740 IF J>1 THEN IF S T ( I , J ) - S T ( I , J - 1 ) < 3 0 0 THEN 3760 3750 GOTO 38203760 PRINT"ERROR. Time p e r i o d m ust be a m u l t i p l e o f 20 s e c o n d s . "3770 PRINT"TIME PERIOD MUST ALSO BE GREATER THAN T = 300 ."3780 PRINT"TIME PERIOD MUST BE AT LEAST 300 SECONDS GREATER THAN

PRECEDING TIME"3790 P R IN T " P E R I O D . : IF J>1 THEN PRINT" LAST TIME PERIOD WAS ==>

" ; S T ( I , J - l ) ; " s e c ." ;C H R $ (1 0 ) ELSE PRINT 3800 GOSUB 1760:GOTO 3670 3810 '3820 NEXT J3830 NEXT I3840 1 SAVE THE DESCRIPTIVE RECORDS3850 '3860 FOR 1=1 TO N3870 GOSUB 2 3 2 0 : ' SET UP DATA FILES3880 GOSUB 2150: 1 OPEN DATA FILE3890 FIELD 1 ,2 AS DU$,2 AS T $ ,236 AS D$3900 '3910 FOR J=1 TO S T S ( I , 1)3920 LSET DU$=MKI$(999)3930 LSET T $ = M K I$ (S T ( l , j ) )3940 LSET D $ = S T $ (I ,J )3950 PUT 1 ,L R (I )3960 L R (I)= L R (I)+ 13970 PRINT I ; L R ( l ) ,3980 NEXT J3990 CLOSE 4000 NEXT I:CLOSE4010 CHAIN MERGE"DUMMY.TXT", 1100, ALL,DELETE 3060-52404020 GOTO 11004030 X$=INPUT$( 1 ) : PRINT X$4040 RETURN

244

4050 1 'PRINT CALIBRATION SPECS4060 PRINT SPC(1 0 ) ; " T ra n s d u c e r # 1 " ;S P C ( 2 0 ) ; " T r a n s d u c e r #

2 " ;C H R $ (1 0 ) :PRINT "Lmax = > ";SPC (9);Y M A X (1),, ;YMAX(2):PRINT"Lmin =>";SPC(9);Y M IN (1),,YM IN(2):PRIN T"V m ax = > " ;SPC( 9 ) ;XMAX( 1 ) , , XMAX( 2 ) : PRINT"Vmin =>";SPC (9);X M IN (1),,X M IN (2);C H R $(10)

4065 RETURN 4070 ’4080 '4090 ' GRAPH LETTERING SETUP ROUTINE4100 FOR I2%-1 TO 184110 FOR J2%=1 TO 74120 READ X :IF X=999 THEN END4130 LT$(l2%)=LT$(l2%)+CHR$(X+32)4140 NEXT J2% ,12%4150 '4160 RETURN4170 '4180 X =A SC (M ID $(L T$(l% ),J% ,l))-32 4190 FOR C%=4 TO 0 STEP -1 4200 IF X>=2',C% THEN 4220 4210 GOTO 4240 4220 GOSUB 5250 4230 X=X-(2~C%)4240 Z=Z-1 4250 NEXT 4260 Y=Y+1 4270 Z=Z+54280 RETURN4290 END4300 OUT 1 2 8 ,1 : OUT 129,Z:OUT 130,Y:RETURN4310 DATA 1 , 1 , 1 , 1 , 1 , 1 , 3 1 : ' L4320 DATA 3 1 , 1 , 1 , 1 5 , 1 , 1 , 3 1 : ' E4330 DATA 1 7 , 1 7 , 1 9 , 2 1 , 2 5 , 1 7 , 1 7 : ' N4340 DATA 1 4 , 1 7 , 1 , 1 , 2 5 , 1 7 , 3 0 : ' G4350 DATA 3 1 , 2 1 , 4 , 4 , 4 , 4 , 4 : ' T4360 DATA 1 7 , 1 7 , 1 7 , 3 1 , 1 7 , 1 7 , 1 7 : ' H4370 DATA 3 1 , 2 1 , 4 , 4 , 4 , 4 , 4 : ' T4380 DATA 1 4 , 4 , 4 , 4 , 4 , 4 , 1 4 : ' I4390 DATA 1 7 , 2 7 , 2 1 , 2 1 , 2 1 , 1 7 , 1 7 : ' M4400 DATA 3 1 , 1 , 1 , 1 5 , 1 , 1 , 3 1 : ' E4410 DATA 1 4 , 1 7 , 2 5 , 2 1 , 1 9 , 1 7 , 1 4 : ' 04420 DATA 4 , 6 , 4 , 4 , 4 , 4 , 1 4 : ' 14430 DATA 1 4 , 1 7 , 1 6 , 1 4 , 1 , 1 , 3 1 : ' 24440 DATA 3 1 , 1 , 1 5 , 1 6 , 1 6 , 1 7 , 1 4 : ' 54450 DATA 2 8 , 2 , 1 , 1 5 , 1 7 , 1 7 , 1 4 : ’ 64460 DATA 8 , 4 , 2 , 2 , 2 , 4 , 8 : ' (4470 DATA 2 , 4 , 8 , 8 , 8 , 4 , 2 : ' )

245

4480 DATA 1 0 , 1 0 , 3 1 , 1 0 , 3 1 , 1 0 , 1 0 : ' #4490 DATA 9994500 X=USR1(1):X=USR0(1)4510 IF GR>0 THEN RETURN 4520 Z=10:Y=62 4530 FOR I%=1 TO 6 4540 GOSUB 5070 4550 Y=Y+6:Z=10 4560 NEXT 1%4570 Z=26:Y=197 4580 I%=11 4590 GOSUB 5070 4600 Y=205:Z=32 4610 GOSUB 5070 4620 '4630 Y=93:Z=26 4640 GOSUB 5070 4650 Y=102:I%=12 4660 GOSUB 5070 4670 Y=0:Z=26 4680 GOSUB 50704690 Z=75:Y=204:FOR I%=1 TO 3 0 :Z=Z+1:GOSUB 5250:NEXT 4700 Y=202:Z=75:FOR L=1 TO 2:FOR I%=1 TO 4:GOSUB

5 2 5 0 :Y=Y+1 : NEXT:Y=2 0 2 :Z=1 0 5 :NEXT 4710 Z=122:Y=205:I%=14:GOSUB 5070 4720 Y=220:Z=120 4730 FOR I%=7 TO 1 0 :GOSUB 5070 4740 Z=Z+8:Y=220 4750 NEXT4760 I%=16:Y=220:Z=1604770 GOSUB 50704780 I%=9:Y=220:Z=1674790 FOR I%=9 TO 8 STEP -14800 GOSUB 50704810 Y=220: Z=Z+8: NEXT4820 Y=205: Z=135: FOR I%=9 TO 8 STEP- 1 : GOSUB 5 0 7 0 :Y=205:Z=Z+8:NEXT4830 I%=3:Y=220:Z=1824840 GOSUB 5070:Y=205:Z=151:GOSUB 50704850 I%=17: Y=220:Z=1884860 GOSUB 50704870 Y=155:Z=7:I%=164880 GOSUB 50704890 Y=155:Z=14:I%=94900 FOR L=1 TO 24910 GOSUB 50704920 Z=Z+7:Y=155:NEXT4930 Y=155:Z=27:I%=174940 GOSUB 5070

246

4950 Y=0:Z=247:I%=184960 GOSUB 50704970 Y=101:Z=2474980 GOSUB 50704990 Y=0:Z=254:I%=125000 GOSUB 50705010 Y=101: Z=254:I%=135020 GOSUB 50705030 Y=200:Z=30:GOSUB 52505040 '5050 15060 RETURN5070 FOR J%=1 TO 7: GOSUB 4180:NEXT 5080 RETURN5090 '5100 1 SET UP GLITCH CATCHER5110 PRINT CHR$(2 6 ) ; SPC( 2 0 ) ; "GLITCH CATCHER ADJUSTMENT";CHR$(10)5120 PRINT"The g l i t c h c a t c h e r a l lo w s t h e p rog ram t o t e l l t h e

d i f f e r e n c e be tw e en "5130 PRINT"a ' r e a l ' v a lu e and an a r t i f a c t u a l change i n t r a n s d u c e r arm

p o s i t i o n . "5140 P R IN T "T y p ic a l ly , t h e ' g l i t c h v a l u e ' i s s e t t o 5% s t a n d a r d e r r o r .

T h i s i s "5150 PRIN T"the d e f a u l t v a l u e . I f t h e d e v i a t i o n in t h e m easu red v a lu e " 5160 PRIN T"is g r e a t e r th a n 5%, t h e com pu te r w i l l i g n o r e t h e v a lu e and" 5170 PRIN T"com pensate f o r t h e ' g l i t c h ' . ";CHR$(10)5180 PRINT"Do you w ish t o change t h e g l i t c h c a t c h e r l e v e l o f g l i t c h

c a t c h i n g (Y/N) ? " ;5190 GOSUB 40305200 IF X$<"Y" THEN 10805210 INPUT"Enter t h e new g l i t c h c a t c h i n g l e v e l (0 .0 0 1 t o 10 % SE)";GLC 5220 IF GLCC.001 OR GLC>10 THEN PRINT"ERROR. TRY AGAIN":G0T0 5210 5225 PRINT"I have a g l i t c h c a t c h e r l e v e l o f " ;G L C ;" % SE":PRINT"Is t h i s

c o r r e c t (Y/N) ":GOSUB 4 0 3 0 : IF X$<"Y" THEN 5210 5230 GOTO 10805240 '5250 ' MATROX GRAPHING ROUTINE5260 OUT 1 2 8 , l:OUT 129,Z:0UT 130,Y:RETURN 5270 ' DISK STORAGE OF CALIBRATION5280 '5290 OPEN " R " , 1 , "CAL.AQU",7 25300 FIELD 1, 4 AS YMAXl$,4 AS YMAX2$,4 AS YMIN1$,4 AS YMIN2$,4 AS

XMAXl$, 4 AS XMAX2$,4 AS XMIN1$,4 AS XMIN2$,4 AS Ml$, 4 AS M2$, 4 AS B l $ , 4 AS B 2$ ,4 AS MVIDYl$,4 AS MVIDY2$,4 AS BVIDY1$,4 AS BVIDY2$,8 AS D$

5310 RETURN 5320 GET 1 , 1 : CLOSE5330 YMAX(1)=CVS(YMAXl$):YMAX(2)=CVS(YMAX2$):YMIN(1)=CVS(YMINl$)>f 5340 YMIN(2)=CVS(YMIN2$) :XMAX(1 )=CVS(XMAXl$) :XMAX(2)=CVS(XMAX2$)

247

5350 XMIN(1 )=CVS(XMIN1$):XMIN(2)=CVS(XMIN2$)5360 M(1)=CVS(M1$):M(2)=CVS(M2$):B(1)=CVS(B1$):B(2)=CVS(B2$)5370 MVIDY( 1 )=CVS(MVIDY1 $ ) :MVIDY( 2)=CVS(MVIDY2$)5380 BVIDY( 1 )=CVS( BVIDY1 $ ) : BVIDY( 2 )=CVS( BVIDY2$)5390 DATE$=D$5400 RETURN5410 ' GLITCH GRABBER5412 IF STER(I,D%)<GLC THEN GLITCH( I)=0:GOTO 54505420 '5445 GLITCH(I)=GLITCH(I)+1 5450 RETURN5460 NUD%=50:GOSUB 6000:NUD%=100 5470 AO( I )=-ABS( A30-ATEMP( I ) )5480 RETURN6000 A 30= 0:S1= 0 :S 2= 0 :FOR C%=1 TO NUD%6010 GOSUB 1560:A30=A30+A:S1=S1+A:S2=S2+(A*A):NEXT 6020 A30=A30/NUD%:GOSUB 1570 6025 A30=A30+1000 6030 RETURN7000 PRINT CHR$(26); : ' Change g l i t c h c a t c h e r l e v e l7010 PRINT "The c u r r e n t g l i t c h c a t c h e r l e v e l i s ==> " ;GLC7020 INPUT"What i s t h e new g l i t c h c a t c h e r l e v e l ";GC7030 IF GLCC.01 OR GLC>30 THEN P R IN T "E rro r . T ry again":GOTO 70107040 PRINT"I have a new g l i t c h c a t c h e r l e v e l o f " ;G C ;" %";CHR$(10)7050 PRINT"Is t h i s c o r r e c t (Y/N) ":GOSUB 40307060 IF X$<"Y" THEN 70107070 GLC=GC:GOSUB 1 8 0 0 :RETURN7080 END18000 PRINT I N P ( 3 0 ) ; I N P ( 3 1 ) , :GOTO 18000

A ppendix I : P rogram L i s t i n g o f D a ta M a n ip u l a t i o n P rog ram , CHANGE/BAS,Used t o A d j u s t D i g i t i z e d D a t a .

T h is p rog ram was w r i t t e n in M i c r o s o f t I n t e r p r e t e r BASIC l i c e n s e d t o R ad io Shack on a TRS-80 m odel I , 48 K, d i s k d r i v e m ic ro c o m p u te r . The o p e r a t i n g sy s te m was DOSPLUS ( v e r s i o n 3 .3 , 3 . 4 ) . T h i s p rogram a l lo w s m o d i f i c a t i o n o f c o o r d i n a t e p a i r s o f numbers from a d i s k f i l e .

1 'CHANGE/BAS V e rs io n 8 .1 ( c ) 1983 by Konrad M. K uzm anoff, a l l r i g h t sreserved

2 '

5 / 4 /8 3

8 '

9 '30 CLEAR 5 0 0 : DEFINT I , J , K , N : DEFSNG X, Y : CMD"F0RMS (P=66,L=54,W =132)" 40 DIM X( 1 0 0 0 ) ,Y(1 0 0 0 ) ,E V $ (100)50 CLS:PRINTCHR$(23);TAB(1 0 ) ; "C hange";CH R$(1 0 ) :PR IN T"(c) 1981 by

Konrad M. Kuzm anoff"5 5 EX=0: FX=0: FV%=0: EV%=0: EC=0: ER=0 60 GOSUB 1 0 0 0 : 1 Get f i l e name70 GOSUB 2 0 8 0 : ' U se r d e f i n e d s t a r t / s t o p p o i n t80 GOSUB 1 1 6 0 : ' Get f i l e o f name FL$90 GOSUB 1410: ' U se r c h o ic e o f a r i t h m e t i c o p e r a t i o n95 IFX>OANDX<8THENGOSUB RANGE 100 ON X GOSUB

1 6 0 0 ,1 7 0 0 ,1 7 8 0 ,1 8 6 0 ,1 9 4 0 ,2 0 1 0 ,3 1 4 0 ,2 2 3 0 ,2 3 9 0 ,2 9 8 0 ,7 0 0 0 ,1 3 0 120 GOTO 90 130 GOSUB 2230 140 '170 CLS:PRINT"BYE."180 END1000 1 File name entry1010 PRINTSTRING$(31,45):PRINT"What i s t h e name":LINEINPUT " o f th e

f i l e ==> ";FL$1020 CLS:PRINT"I have a f i l e name o f ==> ";FL$;CHR$(10)1030 PRINT"Is this correct (Y/N) ?1040 GOSUB 1100 1050 '1060 '1070 IF X$="N" OR X$="n" THEN 50

248

249

1080 PRINTX$:RETURN 1090 '1100 '

1110 ' S i n g l e c h a r a c t e r i n p u t r o u t i n e1120 X$=INKEY$: IF X$="" THEN 11201130 RETURN1140 11150 '1160 ' Get f i l e1170 1 = 1 :CLS: PRINT"Now a c c e s s i n g d a t a f i l e ==> FL$1180 OPEN " I " ,1 ,F L $1190 IF I>2000 THEN 13301200 IF EOF( 1 ) THEN 13101210 INPUT # 1 , X,Y1250 IF FV%>0 AND X<FX THEN 12001260 IF EV%>0 AND X>EX THEN 13101270 X (I)= X : Y (l)=Y1290 PRINT@524,"n = " ; I ;1300 1=1+1:GOTO 1190 1310 N=I-1:CLOSE 1320 RETURN1330 PRINT: PRINT"There a r e more t h a n 1000 v a l u e s . You may";CHR$(10) 1340 PRIN T"(1) Use t h e e x i s t i n g v a lu e s f o r th e SD d e t e r m i n a t i o n . " 1350 PRINT"(2) R e - ru n t h e p r o g r a m ."1360 PRIN T"(3) T e r m in a te t h e p ro g ra m ." ;C H R $ (10)1370 GOSUB 1 1 1 0 : IF VAL(X$)<1 OR VAL(X$)>3 THEN 1370 1380 PRINTX$:0N VAL(X$) GOTO 1 3 1 0 ,1 1 0 0 ,1 3 9 0 1390 CLS:PRINT"BYE.":END 1400 '1410 ' O p t io n l i s t o f a l t e r a t i o n s1420 C L S :P R IN T S T R IN G $ (2 8 ,4 2 )M e n u ";ST R IN G $(28,42)1430 PRINT TAB (7 " 1 . Add t o x o r y . "1440 PRINT TAB (7 " 2. S u b t r a c t from x o r y . "1450 PRINT TAB (7 " 3. M u l t i p l y by a d e s i g n a t e d v a lu e (x o r y)1460 PRINT TAB (7 " 4 . D iv id e by a d e s i g n a t e d v a lu e (x o r y) It•1470 PRINT TAB (7 " 5 . Log ( b a s e 10) o f x o r y . "1480 PRINT TAB (7 " 6. Log ( b a s e e ) o f x o r y . "1490 PRINT TAB (7 " 7 . R e v e r se t h e x , y v a l u e s (x => y , y => x )1500 PRINT TAB (7 " 8 . W r i te t h e changed d a t a t o d i s k . "1510 PRINT TAB (7 " 9. V ideo d i s p l a y l i s t o f t h e d a t a v a lu e s II•

1520 PRINT TAB (7 "1 0 . H ardcopy p r i n t o u t o f d a t a s e t . "1525 PRINT TAB (7 " 1 1 . Go t o more menu c h o i c e s . "1530 PRINT TAB (7 "1 2 . End t h e p ro g ram ." ;C H R $ (1 0 )1540 P R IN T "P lease i n d i c a t e y o u r c h o ic e by num ber. " ; 1560 INPUT X :IF X<1 OR X>12 THEN 1420 1590 RETURN1600 ' Add a number

250

1610 CLS:1620 PRINT"Do you w ish t o add t o t h e x o r y v a lu e s (X/Y) ?1630 GOSUB 1110: IF X$<>"X" AND X$<>"Y" THEN 1610 1640 PRINTX$;CHR$(10)1650 CH=2:IF X$="X" THEN CH=11660 PRINT " P l e a s e e n t e r t h e num ber" :P R IN T "to be added t o t h e " ;X $ ;"

v a l u e s ==> -.INPUT ADD 1670 CLS: PRINTCHR$( 2 3 ) ; "Now a d d i n g " ;ADD; " t o th e ":PRINTX$;"

v a l u e s " ;CHR$(1 0 )1680 GOSUB 2550 1690 RETURN1700 ' S u b t r a c t a number1710 CLS:PRINT"Do you w ish t o s u b t r a c t from th e x o r y v a l u e s (X/Y) ?

i i .1720 GOSUB 1 1 1 0 : IF X$<>"X" AND X$<>"Y" THEN 1710 1730 PRINTX$; CHR$(10)1740 CH=2:IF X$="X" THEN CH=11750 P R IN T "P lease e n t e r t h e number t o be s u b t r a c t e d from t h e

":PR IN TX $;" v a lu e s ==> " ;:INPUT MINUS 1760 CLS:PRINTCHR$( 2 3 ) ; "Now s u b t r a c t in g " ;M IN U S ; " from ":P R IN T "the

" ;X $; " v a lu e s " ;C H R $ (10)1770 GOTO 16801780 1 M u l t i p l y1790 CLS:PRINT"Do you w ish t o m u l t i p l y t h e x o r y v a lu e s (X/Y) ? " ;1800 GOSUB 1 1 1 0 :IF X$<>"X" AND X$<>"Y" THEN1790 1810 PRINTX$ ;CHR$(10)1820 CH=2:IF X$="X" THEN CH=11830 P R IN T "P lease e n t e r t h e number u se d f o r t h e m u l t i p l i e r

o f " :P R IN T " th e " ; X $ ; " v a l u e s ==> INPUT MULTIPLY 1840 CLS:PRINTCHR$(2 3 ) ; "Now m u l t i p l y i n g t h e ";X$:PRINT " v a lu e s by

" ;MULTIPLY;CHR$(10)1850 GOTO 16801860 1 D iv id e1870 CLS:PRINT"Do you w ish t o d i v i d e t h e x o r y v a l u e s (X/Y) ? " ;1880 GOSUB 1 1 1 0 : IF X$<>"X" AND X$<>"Y" THEN18701890 PRINTX$;CHR$(10)1900 CH=2:IF X$="X" THEN CH=11910 P R IN T "P lease e n t e r t h e number u sed f o r t h e d i v i s o r o f " :P R IN T " th e

" ; X $ ; " v a l u e s ==> " ; : INPUT DIVIDE1915 IF DIVIDE=0 P R I N T " I l le g a l d i v i s i o n by ze ro ." :G O T 01910 1920 CLS: PRINTCHR$( 2 3 ) ; "Now d i v i d i n g t h e ";X$:PRINT " v a l u e s by

";DIVIDE;CHR$(10)1930 GOTO 1680 1940 ' LOG1950 CLS:PRINT"Do you w ish t o t a k e th e " :P R IN T " lo g o f t h e x o r y v a l u e s

(X/Y) ?

251

1960 GOSUB 1 1 1 0 : IF X$<>"X" AND X$<>"Y" THEN1960 1970 PRINTX$;CHR$(10)1980 CH=2:IF X$="X" THEN CH=11990 CLS:PRINTCHR$( 2 3 ) ; "Now t a k i n g t h e lo g o f" :P R IN T "th e " ;X $ ;"

v a lu e s ." ;C H R $ ( 1 0 )2000 GOTO 16802010 1 N a t u r a l lo g2020 CLS:PRINT"Do you w ish t o t a k e t h e " : P R I N T " n a t u r a l ' l o g o f

th e" :P R IN T "x o r y v a lu e s (X/Y) ?2030 GOSUB 1 1 1 0 : IF X$<>"X" AND X$<>"Y" THEN2020 2040 PRINTX$;CHR$(10)2050 CH=2:IF X$="X" THEN CH=12060 CLS: PRINTCHR$( 2 3 ) ; "Now t a k i n g th e n a t u r a l lo g " :P R IN T "o f th e

" ; X $ ; " v a lu e s ." ;C H R $ ( 1 0 )2070 GOTO 16802080 ' U se r d e f i n e d s t a r t / s t o p p o i n t2090 CLS: PRINT"You may d e s i g n a t e t h e s t a r t i n g v a lu e t o be u s e d "2100 PR IN T"for t h e c a l c u l a t i o n ( i e . t h e f i r s t v a lu e i n c l u d e d ) . You" 2110 PRINT"may a l s o d e s i g n a t e t h e e n d in g v a lu e ( l a s t v a lu e

i n c lu d e d ) ." ;C H R $ (1 0 )2120 PRINT"Do you w ish t o d e s i g n a t e t h e s t a r t i n g v a lu e (Y/N) ? " ;2130 GOSUB 11102140 PRINTX$;CHR$(10):IF X$="N" OR X$="n" THEN 2180 2150 FV%=9992160 INPUT "What i s t h e s t a r t i n g v a lu e f o r x ";FX 2170 '2180 PRINT"Do you w ish t o d e s i g n a t e t h e e n d in g v a lu e (Y/N) ? " ;2190 GOSUB 11102200 PRINTX$;CHR$(10):IF X$="n" OR X$="N" THEN RETURN 2210 EV%=999:INPUT "What i s t h e e n d in g v a lu e f o r x ";EX 2220 RETURN2230 1 Save c o r r e c t e d d a t a2240 CLS:PRINT"Do you w ish t o s a v e t h e c o r r e c t e d d a t a (Y/N) " ;2250 GOSUB 1110:PRINTX$;CHR$(10):IF X$<"Y" THEN 2380 2253 A%=INSTR(FL$," / " )2255 IF A%=0 THEN FI$=FL$+"/CDT":GOT02260 2257 FI$=LEFT$(FL$,A%-l)+"/CDT"2260 PRINT"Do you w ish t o name t h e f i l e ( d e f a u l t w i l l be ==>

" ; F I $ ; " ) . "2270 P R IN T "P lease answ er (Y/N) ";:G0SUB 11102280 IF X$="Y" THEN PRINT:PRINT " P le a s e e n t e r t h e f i l e name ==>

" ;FI$:LINEINPUTFI$:GOTO 2320 2290 A%=INSTR(FL$ , " / " )2300 IF A%=0 THEN FI$=FL$+"/CDT":GOTO 2320 2310 FI$=LEFT$(FL$,A%—1 )+"/CDT"2320 OPEN " 0 " , 1 ,F I$2330 CLS:PRINT"saving th e d a t a s e t u n d e r t h e name => " ; F I $2340 FOR 1=1 TO N

252

2350 X = IN T (X ( l )* 1 0 0 + .5 ) /1 0 0 :Y = IN T (Y ( l)* 1 0 0 + .5 ) /1 0 02360 PRINT # 1 ,X ; " , " ; Y2370 NEXT2380 CLOSE:RETURN2390 ' L i s t th e v a lu e s2400 CLS:K=12410 PRINT TAB( 2 0 ) ; "The v a l u e s a r e :" ;CH R$(10)2420 FOR 1=1 TO N STEP 2 2430 FOR J= 0 TO 1 2440 IF I+J>N THEN 2520 2450 PRINTCHR$(123);2460 PRINT USING " # # # # .# # " ; X ( J + I ) ;2470 PRINTUSING " # # # # # .# # # " ; Y ( J + I ) ;2480 PRINTCHR$( 1 2 5 ) ; STRING$( 6 , 3 2 ) ;2490 NEXT:PRINT:K=K+12500 IF K>10 THEN PR IN T"Press any key t o c o n t i n u e . ";:GOSUB 1 1 1 0 : IF

X$="R" THEN 90 ELSE CLS:K=0:2520 NEXT2530 PRINT:PRINT T A B (4 0 );"n = ";N 2535 GOSUB3230 2540 RETURN2550 ' A l t e r a t i o n o f v a lu e s2560 ON CH GOTO 2590 ,2740 2570 '2580 '2590 ' A l t e r x v a l u e s2600 FOR I=lTON2605 IF X( I )< SVORX( I ) >LVTHE NAGAIN 2610 POKE 15422 ,422620 ON X GOTO 2 6 3 0 ,2 6 4 0 ,2 6 5 0 ,2 6 6 0 ,2 6 7 0 ,2 6 9 02630 X( I )= X ( I)+ADD: GOTO 27202640 X(l)=X(l)-MINUS:GOTO 2720-2650 X( I )= X ( I)*MULT:GOTO 27202660 X(I)=X(I)/DIV:GOTO 27202670 IF X (I)< = 0 THEN ER=999:GOTO 27102680 X(I)=LOG(X(I))/LOG(10):GOTO 27202690 IF X (I)< = 0 THEN ER=999:GOTO 27102700 X (l)= L O G (X (l) )2710 IF ER=999 THEN GOSUB2890 2720 POKE 15422 ,79 2725 NAME AGAIN: NEXT I2730 RETURN2740 ' A l t e r y v a lu e s2750 FOR 1=1 TO N2755 IFX(I)<SVORX(l)>LVTHEN GOTO AGAIN 2760 POKE 15422 ,422770 ON X GOTO 2 7 8 0 ,2 7 9 0 ,2 8 0 0 ,2 8 1 0 ,2 8 2 0 ,2 8 4 0 2780 Y( I )= Y ( I)+ADD:GOTO 2870

253

2790 Y(I)=Y(I)-M INUS:G0T0 28702800 Y(l)=Y(l)*MULT:GOTO 28702810 Y(l)=Y(I)/DIV:GO TO 28702820 IF Y (I)< = 0 THEN ER=999:GOTO 28602830 Y(I)=LOG(Y(I))/LOG(10):GOTO 28702840 IF Y (I)< = 0 THEN ER=999:GOTO 28602850 Y (I )= L 0 G (Y (I ) )2860 IF ER=999 THEN GOSUB2890 2870 POKE 1 5 4 2 2 ,3 2 :NEXT 2880 RETURN2890 ' C a n ' t do i t . E x c lu d e t h e v a lu e 2900 EC=EC+12910 E V $(E C )= S T R $ (X (l) )+ " ,"+ S T R $ (Y (l) )2920 PR IN T @ 256 ," Inva lid o p e r a t io n " :P R I N T " lo g ( v a l u e <= 0 ) . "2925 PRINT "Must e x c lu d e " :P R IN T " th e v a lu e ==> ";EV$(EC)2930 FOR J= I+ 1 TO N2940 X ( J - l ) = X ( J ) :Y ( J - 1 ) = Y (J )2950 NEXT 2960 N=N-1:1=1-1 2965 ER=02967 PRINT@256,CHR$( 3 0 ) ; : PRINTCHR$( 3 0 ) : PRINTCHR$( 3 0 ) : PRINTCHR$(30) 2970 RETURN2980 ' H ardcopy l i s t i n g2990 CLS:PRINT"You have r e q u e s t e d a h a rd c o p y l i s t i n g . " ; C H R $ ( 1 0 ) 3000 P R IN T "P repare y o u r p r i n t e r ( a d j u s t to to p o f form ,

e t c . ) . " ; C H R $ ( 1 0 )3010 PR IN T"If you d o n ' t h ave a p r i n t e r r e a d y t o r e c e i v e t h e

d a t a , " :P R I N T " th e n p r e s s 'R ' t o r e t u r n t o t h e menu. "3020 GOSUB 3220 3030 IF X$="R" THEN RETURN 3040 CLS:PRINT"***************** PRINTING

***** * * * * * * * * * * * " ;CHR$( 1 0 ) ;CHR$(10)3050 LPRINT TAB(1 0 ) ; "D a ta s e t i s f i l e name ==> ";FL$;CHR$(10)3055 LPRINT TAB(30);TIME$;CHR$(10)3060 FOR 1=1 TO N 3070 LPRINT CHR$(123);3080 LPRINT USING " # # # # .# # " ; X ( l ) ;3090 LPRINT3100 LPRINT USING " # # # # .# # " ; Y ( I ) ;3110 LPRINT CHR$(125),3120 NEXT3130 LPRINTCHR$( 1 0 );C H R$(1 0 ) : LPRINT "n = " ;N;CHR$( 1 2 ) ; : RETURN3140 1 R e v e r s e t h e x , y v a lu e s3150 CLS:PRINTCHR$(2 3 ) ; " R e v e r s in g t h e x , y v a l u e s . "3160 FOR 1=1 TO N 3170 POKE 15422 ,42 3180 T = X ( I ) :X ( I ) = Y ( I ) :Y ( I ) = T 3190 POKE 15422 ,32

3200 NEXT 3210 RETURN3220 ' P au se c o n t r o l3230 PRINT: PR IN T"Press any key t o c o n t i n u e . GOSUB

1 1 1 0 :PRINTX$:RETURN 7000 ' More menu commands7010 C L S :P R IN T S T R IN G $ (2 8 ,4 2 )M e n u 2 ";STRING$(27, 4 2 ) ;CHR$(10) 7020 PRINT TA B(10)"1. R e tu rn t o main m en u ."7030 PRINT TA B(10)"2 . Examine and a l t e r a n o t h e r d a t a s e t . "7095 PRINT TA B(10)"3 . R e tu rn t o m ain DOS m enu." ;CH R $(10)7100 P R IN T "P lease e n t e r yo u r c h o ic e by num ber. " ;7110 INPUT X: IF X<1 OR X>9 THEN 7000 7120 ON X GOTO 1 4 2 0 ,5 0 ,7 1 3 0 7130 RUN"MENU/BAS"7190 END8000 NAME RANGE: 1 CHOOSE START AND STOP OF VALUES 8010 CLS:PRINTTAB(31)"Menu";CHR$(10);CHR$(10)8020 PRINTTAB(5)"1. D e s ig n a te s t a r t i n g x v a lu e f o r m a n i p u la t i o n 8030 PRINTTAB(5)"2. D e s ig n a t e LAST v a l u e f o r x ( s t a r t a t

b e g i n n i n g ) "8040 PRINTTAB(5)"3. D e s ig n a te BOTH s t a r t i n g and e n d in g v a l u e " 8050 PRINTTAB( 5 ) " 4 . Use t h e w hole f i l e " ;C H R $ ( 1 0 )8060 SV=X(1):LV=X(N):INPUT@ 448,"Please e n t e r y o u r c h o ic e by numbe

" , 1 G$: J J=VAL( G$)8070 CLS:0N JJGOSUB START,LAST,BOTH,EXIT: RETURN 8080 NAME EXITrRETURN8090 NAME BOTH:1 s e t b o th s t a r t and s to p 8100 GOSUB START: GOSUB LASTrRETURN 8110 NAME START8120 IN PU T"Please e n t e r t h e s t a r t i n g x v a l u e ";SV 8130 RETURN 8140 NAME LAST8150 IN PU T"Please e n t e r t h e e n d in g x v a lu e ";LV 8160 RETURN

A ppendix J : P rogram L i s t i n g o f L i n e a r R e g r e s s i o n P ro g ra m , LINREG/BAS,Used t o C a l c u l a t e B e s t F i t S t r a i g h t , L o g a r i t h m i c , arid E x p o n e n t i a l C u r v e s .

T h is p ro g ram was w r i t t e n in M i c r o s o f t I n t e r p r e t e r BASIC l i c e n s e d t o R ad io Shack on a TRS-80 m odel I , 48 K, d i s k d r i v e m ic ro c o m p u te r . The o p e r a t i n g sy s te m was DOSPLUS ( v e r s i o n 3 . 3 , 3 . 4 ) .

10 OUT 95 ,120 'L i n e a r R e g r e s s i o n C a l c u l a t i o n40 ' ( C ) 1983, by Konrad M. K uzm anoff, a l l r i g h t s r e s e r v e d . V e r s io n

3 . 1 , 3 /1 5 /8 3 60 DEFINT I , J , K , N , L : DEFSNG X,Y,S80 DIM X (1 0 0 0 ) ,Y ( 1 0 0 0 ) ,EV$( 2 0 0 ) ,R ( 3 ) ,M ( 3 ) ,B ( 3 ) ,E Q $ ( 3 ) ,N (3 )100 E Q $ ( l )= " y = mx + b " :E Q $ (2 )= "y = A e x p (k x ) w i t h k = m, A = e x p (b ) " :E Q $ (3 ) = " y = A l o g ( x ) +b w i t h A = m"120 C LS:PRIN TC H R$(23);TA B(5);"L inear R e g r e s s i o n

F i t" ;C H R $ (1 0 ) :P R IN T " (c ) 1981, by Konrad M. Kuzm anoff"140 K=1:FV%=0:EV%=0:FX=0:EX=0160 GOSUB 4 0 0 : ' Get f i l e name180 GOSUB 1 2 2 0 : ' U se r d e f i n e d s t a r t / s t o p p o i n t200 GOSUB 7 2 0 : 1 Get f i l e o f name FL$220 FOR L=1 TO 3240 GOSUB 1 8 6 0 : ' C a l c u l a t e l i n e a r r e g r e s s i o n c u rv e260 GOSUB 2 8 4 0 : ' P a u se f o r u s e r280 GOSUB 2 5 2 0 : 1 D i s p la y o f v a l u e s320 NEXT L340 GOSUB 3 2 0 0 : ' MENU360 CLS:PRINT"BYE."380 END400 ' F i l e name e n t r y420 PRINTSTRING$( 3 1 , 4 5 ) :PRINT"What i s t h e name":LINEINPUT"of t h e f i l e

==> " ;FL$440 CLS: PRINT"I have a f i l e name o f ==> ";FL$;CHR$(10)460 PRINT"Is t h i s c o r r e c t (Y/N) ?480 GOSUB 600 500 '520 '540 IF X$="N" OR X$="n" THEN 400 560 PRINTX$:RETURN620 1 S i n g l e c h a r a c t e r i n p u t r o u t i n e

255

256

640 X$=INKEY$: IF X$="" THEN 640 660 RETURN 680 '700 1720 ' Get f i l e740 1 = 1 :CLS: PRINT"Now a c c e s s i n g d a t a f i l e ==> FL$760 OPEN " I " , 1 ,FL$780 IF I>1000 THEN 1060800 IF E 0F (1 ) THEN 1020820 INPUT # 1 , X,Y900 IF FV%>0 AND X<FX THEN 800920 IF EV%>0 AND X>EX THEN 1020940 X (I)= X : Y(I)=Y980 PRINT@524,"n = " ; I ;1000 1= 1+ 1 :GOTO 780 1 0 2 0 .N=I-1:CLOSE 1040 RETURN1060 PRINT:PRINT"There a r e more t h a n 1000 v a l u e s . You may";CHR$(10) 1080 PR IN T"(1) Use t h e e x i s t i n g v a l u e s f o r th e SD d e t e r m i n a t i o n . " 1100 PR IN T"(2) R e - ru n t h e p r o g ra m ."1120 PR IN T"(3) T e r m in a te t h e p ro g ra m ." ;C H R $ (10)1140 GOSUB 6 2 0 : IF VAL(X$)<1 OR VAL(X$)>3 THEN 1140 1160 PRINTX$:ON VAL(X$) GOTO 1 0 2 0 ,6 0 0 ,1 1 8 0 1180 CLS:PRINT"BYE.":END 1200 '

1220 ' U se r d e f i n e d s t a r t / s t o p p o i n t1240 CLS:PRINT"You may d e s i g n a t e t h e s t a r t i n g v a l u e t o be u s e d "1260 PR IN T"for t h e c a l c u l a t i o n ( i e . t h e f i r s t v a l u e i n c l u d e d ) . You" 1280 PRINT"may a l s o d e s i g n a t e t h e e n d in g v a l u e ( l a s t v a lu e

i n c l u d e d ) . " ;C H R $ ( 10)1300 PRINT"Do you w ish t o d e s i g n a t e t h e s t a r t i n g v a lu e (Y/N) ? " ;1320 GOSUB 6201340 PRINTX$;CHR$(10):IF X$="N" OR X$="n" THEN 1420 1360 FV%=9991380 INPUT "What i s t h e s t a r t i n g v a lu e f o r x ";FX 1400 '1420 PRINT"Do you w ish t o d e s i g n a t e t h e e n d in g v a l u e (Y/N) ? " ;1440 GOSUB 6201460 PRINTX$;CHR$(10):IF X$="n" OR X$="N" THEN RETURN1480 EV%=999:INPUT "What i s t h e e n d in g v a l u e f o r x ";EX1500 RETURN1520 1 Save c o r r e c t e d d a t a1540 CLS:PRINT"Do you w ish t o s a v e th e e q u a t i o n s (Y/N)?1560 GOSUB 6 2 0 : IF X$<"Y" THEN 18401580 A%=INSTR(FL$," / " ) : IF A%=0 THEN 1620 1600 FI$=LEFT$(FL$,A%-l)+"/CDT"1620 PRINT"Do you w ish t o name t h e f i l e ( d e f a u l t w i l l be ==>

" ; F I $ ; " ) . "

257

1640 P R IN T "P lease answ er (Y/N) ";:GOSUB 6201660 IF X$<>"N" AND X$<>"n" THEN INPUT " P le a s e e n t e r t h e f i l e name ==>

";FI$:GOTO 1740 1680 A%=INSTR(FL$, " / " )1700 IF A%=0 THEN FI$=FL$+"/CDT":GOTO 1740 1720 FI$=LEFT$(FL$,A%-l)+"/CDT"1740 OPEN " 0 " , 1 ,F I$1760 FOR I=N TO 11780 D$=STR$(X(I) ) + " , "+S T R $(Y (I))1800 PRINT # 1 , D$1820 NEXT1840 CLOSE:RETURN1860 ' L i n e a r R e g r e s s i o n A n a ly s i s1880 CLS:PRINT" C a l c u l a t e L i n e a r R e g r e s s i o n F i t #";L ;CH R$(10)1900 S 1 = 0 :S 2 = 0 :S3=0: S4=0: S5=0: N2=N 1920 FOR 1=1 TO N 1940 ON L GOTO 1 9 6 0 ,1 9 8 0 ,2 0 2 0 1960 X=X(l):Y=Y(l):GOTO 20401980 X=X( I ) : IF Y ( l )> 0 Y=L0G (Y (l)) ELSE N=N-l:GOTO 2180 2000 GOTO 20402020 Y=Y( I ) : IF X ( l )> 0 THEN X=LOG(X(l)) ELSE N=N-1:GOTO2180 2040 POKE 1 5 4 2 0 ,42:POKE15422,32 2060 S1=S1+X 2080 S2=S2+(X*X)2100 S3=S3+Y 2120 S4=S4+(X*Y)2140 S5=S5+(Y*Y)2160 POKE 1 5 4 2 0 ,32:POKE 15422 ,42 2180 NEXT2190 IF N<=0THEN PRINT"I c a n ' t c a l c u l a t e f o r l o g ( ) value":GOTO 2320 2200 M (L )= ( (S 1 * S 3 /N ) -S 4 ) / ( (S 1 * S 1 /N ) -S 2 )2220 B (L )= (S 3 /N )-M (L )* (S 1 /N )2240 SX=( ( S 2 /N ) - ( ( S1*S1 ) / (N*N) ) ) * ( N / ( N - l ) ) : SX=SQR( SX)2260 S Y = ((S 5 /N )- ( (S 3 * S 3 ) /(N * N )) )* (N /(N -1 ) ) :S Y = S Q R (S Y )2280 R(L)=M(L)*SX/SY 2300 N(L)=N2320 PRINT"Using th e g e n e r a l e q u a t i o n ==> " ;EQ $(L);CH R$(10)2340 PRINT"With n = " ;N (L )2360 PRINT:PRINT"m = " ;2380 PRINTUSING " # # # # # # .# # # # " ;M(L)2400 PRINT"b = " ;2420 PRINTUSING " # # # # # # .# * # # " ;B(L)2440 P R I N T " c o r r e l a t i o n c o e f f i c i e n t =2460 PRINTUSING " # # .# # # # " ; R(L)2480 N(L)=N:N=N2 2500 RETURN2520 ' D i s p l a y v a lu e s2540 CLS:PRINT"Do you w ish t o v iew th e d a t a s e t s u se d (Y/N) ? " ;

258

2560 GOSUB 6 2 0 : IF X$="Y" THEN 26002580 RETURN2600 CLS:J=12620 FOR 1=1 TO N2640 ON L GOTO 2 6 6 0 ,2 6 8 0 ,2 7 2 02660 X=X(l):Y=Y(l):GOTO 27402680 X=X( I ) : IF Y ( l ) > 0 THEN Y=LOG(Y(I)) ELSE 2800 2700 GOTO 27402720 Y=Y( I ) : IF X ( l ) > 0 THEN X=LOG(X(l)) ELSE 2800 2740 PRINTCHR$(123);X;" , " ;Y ;C H R $(125 ); "2760 IF POS(0)>40 THEN J=J+1:PRINT2780 IF J>10 THEN GOSUB 2 8 4 0 : IF X$="R" THEN RETURN ELSE CLS:J=1 2800 NEXT 2820 RETURN2840 PRINT: PR IN T"Press any k ey t o c o n t i n u e . "2860 GOSUB 620: RETURN2880 CLS :PRINTTAB(1 0 ) ; "V a lues a r e :" ;CH R$(10)2900 GOTO 28002920 ' H ardcopy2940 CLS: P R IN T "P lease p r e p a r e you r p r i n t e r . I f no p r i n t e r i s

p r e s e n t , "2960 PR IN T"then p r e s s ' R ' . P r e s s any k ey when r e a d y . " ;2980 GOSUB 6 2 0 : IF X$="R" THEN RETURN 3000 FOR L=1 TO 33010 LPRINT "F or t h e d a t a s e t f i l e ==> ";FL$3020 LPRINT "U sing t h e g e n e r a l e q u a t i o n = = > ";E Q $(L );C H R $(10): LPRINT

"W ith n = " ;N ( L ) : LPRINT "m = " ;M(L):LPRINT "b = " ;B ( L ) : LPRINT " t h e c o r r e l a t i o n c o e f f i c i e n t r =";R(L):LPRINT

3140 NEXT3160 LPRINT CHR$(12);3180 RETURN3200 ' MENU3220 CLS:PRINTSTRING$(28,4 2 ) ; " Menu " ;ST R IN G $(28,42)3240 PRIN T"(1) H ardcopy o u t p u t o f c a l c u l a t e e q u a t i o n s "3260 PRIN T"(2) D i s p la y c a l c u l a t e d e q u a t i o n s .3280 PRIN T"(3) D i s p l a y v a l u e s u s e d f o r c a l c u l a t i o n s . "3300 PRINT"(4) Exam ine a n o t h e r d a t a s e t . "3320 PRIN T"(5) R e tu r n t o m ain DOS m enu ."3340 PRIN T"(6) End t h e p ro g ra m . T e r m in a te a l l d a t a

m a n ip u la t io n s ." ;C H R $ (1 0 )3360 PR IN T "P lease e n t e r y o u r c h o ic e by num ber. " ;3380 INPUT X :IF X<1 OR X>6 THEN 32203400 ON X GOSUB 2920 ,3*51073460 ,120 ,3480 ,11803420 GOTO 32003440 FOR L=1T03:CLS: GOSUB 2320:GOSUB 2 8 4 0 :NEXT:GOTO 3200 3460 FOR L=1 TO 3 : CLS:PRINT"Using ==> " ;E Q $ (L ) : GOSUB

2 8 4 0 :GOSUB2600:GOSUB 2 8 4 0 :NEXT:GOTO 3200 3480 RUN"MENU/BAS"

A ppend ix K: P rogram L i s t i n g o f P o ly n o m ia l C urve F i t t i n g P rogram ,MATRIX2/BAS.

T h is p ro g ra m was w r i t t e n i n M i c r o s o f t I n t e r p r e t e r BASIC l i c e n s e d t o R a d io Shack on a TRS-80 m odel I , 48 K, d i s k d r i v e m ic ro c o m p u te r . The o p e r a t i n g sy s te m was DOSPLUS ( v e r s i o n 3 . 3 , 3 . 4 ) .

10 CLEAR 50040 CLS: PRINTCHR$( 2 3 ) ; STRING$( 1 0 , 3 2 ) ; "CURVE F IT "5051

MATRIX2 / BAS

(C) 1983 BY KONRAD M. KUZMANOFF525354 ' VERSION 9 .1 060 DEFDBL A ,B ,C ,S ,X ,Y : DEFINT I , J ,K ,M80 DIM X ( 1 5 0 ) ,Y ( 1 5 0 ) ,C ( 1 2 ) ,A ( 1 2 ,1 3 ) ,X N ( 1 5 0 ) ,B T ( 1 2 ) ,S $ ( 1 2 ) ,E $ ( 1 2 )90 B$ -"# # # # # # # .# # # # # C$ « " # # # .# # # # "100 FOR 1=0 TO 1 50 :X (I)= 0 :Y (I)= 0 :X N (I)= 0 :N E X T110 FOR 1=0 TO 12: B T ( l ) = 0 : S $ ( l ) = " " : C (I)=0 :FO R J= 0 TO 13:

A(l,J)=0:NEXT:NEXT 115 FOR 1=0 TO 12 :E$(I)="" :N EX T 120 1 = 0 :J=0:HC=0140 CLS: INPUT"DO YOU WISH A HARD COPY LISTING (Y/N ENTER)";G$150 IF G$>"N" THEN HC=999160 GOSUB 1700 :PRINT "YOU HAVE TWO CHOICES":PRINT CHR$( 2 0 0 ) ; "CHOICE

# 1 " : PRINT "DO YOU WISH TO ENTER DATA FROM":INPUT "THE KEYBOARD (Y/N ENTER)";G$

170 IF G$>"N" THEN GOSUB 1720 : CLS: GOTO 250180 PRINT CHR$(200);"CHOICE #2":PRINT "DO YOU WISH TO ENTER DATA

FROM":INPUT "A USER FILE (Y/N ENTER)";G$190 IF G$>"N" THEN GOSUB 1890 :GOTO 240 200 GOTO 140 240 GOSUB 1760250 CLS:INPUT"WHAT IS THE MINIMUM POLYNOMIAL DEGREE ";MS 260 PRINT"WHAT IS THE MAXIMUM POLYNOMIAL DEGREE":PRINT "IT MUST BE

LESS THAN ==> 13":INPUT MF 270 'CHECK IF MAX DEGREE (MF) IS LARGER THAN N -l 280 IF MF<=12 THEN 320 290 MF=12300 PRINT "THE DEGREE OF THE HIGHEST POLYNOMIAL EXCEEDS 12";310 PRINT "THE HIGHEST DEGREE IS NOW ==> 12"320 Ml=MF+l 330 M2=MF+2

259

260

340 FOR 1=1 TO N 350 XN(I)=1 360 NEXT I370 CLS:PRINT@320,"COMPUTING THE MATRIX OF SIMULTANEOUSEQUATIONS":'COMPUTE 1ST AND N+l COLUMN OF A. I MOVES DOWN ROWS, J SUMSOVER THE N VALUES380 FOR 1=1 TO Ml390 A ( I ,1 ) = 0400 A (I ,M 2)=0410 FOR J=1 TO N420 A (I ,1 )= A (I ,1 )+ X N (J) :P 0 K E 1 5 3 6 0 ,79:POKE 15380 ,42430 A (I ,M 2)= A (I ,M 2)+ Y (J)*X N (J)440 XN (J)=XN (J)*X(J):P0KE 1 5 3 6 0 ,4 2 :POKE 15380 ,79450 NEXT J460 NEXT I480 PRINT@320,STRING$(60,3 2 ) ;:PRIN T@320,"CALCULATE THE Y VALUES "490 FOR 1=2 TO Ml500 A(M1, I ) = 0510 FOR J=1 TO N520 A (M 1,I)=A (M 1,I)+X N (J)530 POKE 1 5 3 6 0 ,79:POKE 15380 ,42 540 X N (J )= X N (J)* X (j)550 POKE 1 5 3 6 0 ,42:POKE 15380 ,79 560 NEXT J570 NEXT I 580 CLS600 FOR J= 2 TO Ml 610 FOR 1=1 TO MF620 A ( l , J ) = A ( I + 1 , J - 1 )630 NEXT I640 NEXT J650 PRINT"SOLVE THE MATRIX OF SIMULTANEOUS EQUATIONS"670 GOSUB 1270 690 MP=MS+1 700 FOR I=MP TO Ml710 FOR J=1 TO I720 C (J)= A (J ,M 2)730 NEXT J750 GOSUB 1490760 IM=I-1780 PRINT"FOR D E G R E E IM ; "THE COEFFICIENTS ARE"790 IF HC>0 THEN LPRINT CHR$( 1 0 ) ; "FOR D E G R E EIM ; "THECOEFFICIENTS ARE :":PRINT800 FOR J=1 TO I810 PRINT USING C $ ; C ( j ) ,820 IF HC>0 THEN LPRINT C ( j )830 E $ ( l)= E $ ( l)+ M K D $ (C (J ))840 NEXT J

261

850 PRINT 870 BETA=0880 FOR IP=1 TO N890 SUM=0900 FOR IC0EF=2 TO I910 JC0EF=I-IC0EF+2920 SUM=(SUM+C( JCOEF) ) *X( I P )930 NEXT ICOEF940 SUM=SUM+C(1)950 BETA =(Y (IP)-SU M )*(Y (IP)-SU M )960 NEXT IP970 BETA = BETA/(N-(1 - 1 ) )980 PRINT"CORRELATION IS ==> 1-BETA990 IF HC>0 THEN LPRINT:LPRINT "CORRELATION IS ==> " ; 1-BETA1000 BT(IM)=BETA 1010 NEXT I1100 IB=MS:BA=BT(MS):FOR I=MS TO MF: IF ABS(BA)>ABS(BT(l)) THEN B A =B T (l) :IB =I 1110 NEXT1120 PRINT:PRINT"HIT ENTER TO CONTINUE " : GOSUB 30001130 CLS:PRINT"THE POLYNOMIAL OF DEGREE " ; I B IS THE BEST FIT "1140 PRINT:PRINT"THIS EQUATION IS : "1150 IB=IB+11160 FOR 1=1 TO L E N (E $(IB )) STEP 81170 SV=CVD(MID$(E$(IB), 1 , 8 ) ) : PRINT SV; " X [ " ; ( l - l ) / 81180 IF HC>0 THEN LPRINT CVD(MID$(E$(IB), 1 , 8 ) ) ; " X [ " ; ( I - l ) / 81190 NEXT I1200 OUT 2 5 4 ,01210 GOSUB 21001220 PRINT:PRINT "DO YOU WISH TO ANALYSE ANOTHER SET OF DATA (Y/N ENTER)1225 GOSUB 30001230 IF G$>"N" THEN GOTO 1001240 CLS:PRINT"DO YOU WISH TO PLOT THIS DATA (Y/N ENTER)";1245 GOSUB 30001250 IF G$>"N" THEN RUN"PLOT/BAS"1260 CLS:PRINT"BYE":END 1270 '1280 FOR 12=1 TO Ml1290 FOR J2= 2 TO Ml 1300 SUM=01310 IF J2> 12 THEN 13801320 JM=J 2-11330 FOR K=1 TO JM1340 SUM=SUM+A(I2,K)*A(K,J2)1350 NEXT K1360 A ( I2 ,J 2 )= A ( I2 ,J 2 ) -S U M1370 GOTO 1450

262

1380 IM=I2-11390 IF IM=0 THEN 14301400 FOR K=1 TO IM1410 SUM=SUM+A(I2,K)*A(K,J2)1420 NEXT K1430 IF A B S (A (I2 ,I2 ) )< 1 E -1 0 THEN 14701440 A ( I 2 , J 2 ) = ( A ( I 2 , J 2 ) - S U M ) / A ( I 2 , I 2 )1450 NEXT J 2 , I 21460 RETURN1470 PRINT"REDUCTION INCOMPLETE. VALUE IN RO W ";I2 ;" IS TOO SMALL"1480 GOTO 18801490 'THIS SUBROUTINE SOLVES N LINEAR EQUATIONS 1500 C (1 )= C (1 ) / A ( l , 1 )1510 FOR 12=2 TO I 1520 IM=I2-1 1530 SUM=01540 FOR K=1 TO IM1550 SUM=SUM+A(I2,K)*C(K)1560 NEXT K1570 C ( I 2 ) = ( C ( I 2 ) - S U M ) /A ( I 2 , I 2 )1580 NEXT 12 1590 'BACK SUBSTITUTE 1600 FOR J2= 2 TO I 1610 N 2=I-J 2+2 1620 N 1 = I -J 2+1 1630 SUM=01640 FOR K=N2 TO I1650 SUM=SUM+A(N1,K)*C(K)1660 NEXT K1670 C(N1)=C(N1)-SUM 1680 NEXT J 2 1690 RETURN1700 CLS: PRINT CHR$( 2 3 ) ;STRING$(1 0 , 3 2 ) ; "CURVE F I T " :PRINT"THIS PROGRAM FITS POLYNOMIALS TOUSER PROVIDED DATA"1710 RETURN1720 INPUT"ENTER THE NUMBER OF DATA SETS";N:FOR 1=1 TO N: PRINT"WHAT IS THE VALUE OF X FOR DATA SET # " ; I 1730 INPUT X ( I )1740 INPUT "WHAT IS THE Y VALUE";Y(l)1750 CLS:NEXT1760 CLS:PRINT"I HAVE THE FOLLOWING VALUES 1770 FOR 1=1 TO N1780 LL=PO S(0)+LEN (STR$(X (l)))+LEN (STR$(Y (l)))+5 1790 IF LL>63 THEN PRINT1800 PRINT I ; " : " ; X ( I ) " , " ; Y ( I ) ; STRING$(3,3 2 ) ;1810 NEXT1820 PRINT:PRINT"IF YOU WISH TO MAKE ANY CORRECTIONS":PRINT"PLEASE ENTER THE NUMBER FOR THE DATA SET TO BE CORRECTED":INPUT"ENTER 0 FOR

263

1830 IF C0=0 THEN 21951840 PRINT"WHAT IS THE CORRECT X VALUE FOR DATA SET CO:INPUT X(CO) 1850 INPUT "WHAT IS THE CORRECT Y VALUE";Y(CO)1860 CLS: GOTO 17701870 STOPrFOR W=0 TO 2 0 0 :NEXT:RETURN1880 END1890 '1900 'READ A USER CREATED FILE 1910 CLS1920 INPUT"ENTER THE NAME OF THE DATA FILE TO BE ANALYZED";FL$1930 CLS1940 PRINT"I HAVE A FILE NAME OF ===> ";FL$1950 INPUT"IS THIS CORRECT (Y/N ENTER)";G$1960 IF G$<"Y" THEN 1990 1970 '1980 OPEN " I " , 1 ,FL$1990 IF E O F (l) THEN 2080 2000 1= 1+1 2010 LINE INPUT #1,D$2020 OUT 254 ,1 2030 A%=INSTR(D$, " , " )2040 X(I)=VAL(LEFT$(D$,A%-1))2050 Y( I)=VAL(RIGHT $ ( D$, LEN( D$) -A%) )2070 GOTO 19902080 CLOSE:N=I:RETURN2090 ' STORE THE ANALYSIS2100 FI$="EQU/AN"2110 OPEN " R " , 1 , F I $ : FIELD 1 ,1 2 AS F $ ,2 AS Q $,2 AS W$,2 AS P $ ,2 3 7 AS EQ$2120 LSET F$=FL$2130 LSET Q$=MKI$(IB)2140 LSET W$=MKI$(MS)2150 LSET P$=MKI$(MF)2160 LSET EQ$=E$(IB)2170 PUT 1 ,1 : CLOSE2180 IF HC>0 THEN LPRINT:LPRINT "DATA ANALYSIS IS NOW STORED UNDER":LPRINT "FILE NAME ==> EQU/AN"2190 RETURN2195 IF HC<999 THEN 22502200 CLS:INPUT"PREPARE PRINTER FOR DATA. HIT ENTER WHEN READY";G$2210 LPRINT STRING$( 1 0 , 3 2 ) ; "DATA TO BE ANALYZED"2220 FOR 1=1 TO N2230 LPRINT I ; " : " ; X ( l ) ; Y ( l )2240 NEXT: LPRINT 2250 RETURN3000 G$=INKEY$: IF G$="" THEN 3000 3010 RETURN

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