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ZEITSCHRIFT FÜR N A T U R F O R S C H U N G
SECTION C
B I O S C I E N C E S
Council
E . BÖNNING, Tübingen A. BUTENANDT, München M. E I G E N ,
Göttingen
Editorial Board
A. HÄGER, Tübingen W. HASSELBACH, Heidelberg P. KARLSON, Marburg
F. KAÜDEWITZ, München E. W E C K E R , Würz bürg
Advisory Editorial Board P. BÖGER, Konstanz D . BÜCKMANN, Ulm K
. G. GÖTZ, Tübingen G. GOTTSCHALK, Göttingen H . HOFFMANN-BERLING,
Heidelberg R . JAGNICKE, Regensburg G. F. M E Y E R , TÜ Inngen M .
R A J E W S K T , Essen
H . SCHIMASSEK, Heidelberg D . SCHULTE-FROHLINDE, Mülheim/R F .
F . SEELIG, Tübingen J . SEELIG, Basel H . SIMON, München W.
STBGLICH, Bonn A. TREBST, Bochum
EDITED IN COLLABORATION
WITH THE INSTITUTES OF THE MAX-PLANCK-GESELLSCHAFT
V O L U M E 37c N U M B E R 1/2 J A N U A R Y / F E B R U A R Y
1982
V E R L A G D E R Z E I T S C H R I F T F Ü R N A T U R F O R S
C H U N G
T Ü B I N G E N
1 W
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Anschrift des Verlages: Postfach 26 45, D-74 Tübingen Satz und
Druck: Konrad Triltsch, Wurzburg
Nachdruck — auch auszugsweise — nur mit schriftlicher
Genehmigung des Verlags
Bayerisch« j Staatsbibliothek I
München J
Section a Physics, Physical Chemistry, Cosmic Physics
Section b Inorganic and Organic Chemistry
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Volume 37 c Zeitschrift für Naturforschimg 1982
Conten t s
Con ten t s of N u m b e r 1/2
O r i g i n a l C o m m u n i c a t i o n s
Antibiotics from Basidiomycetes. X V [1].
1-Hydroxy-2-nonyn-4-one, an Antifungal and Cytotoxic Meta-bolite
from I s c h n o d e r m a benzoinum (Wahl.) Karst. (In German) T.
A N K E , B . - M . G I A N N E T T I , and W. S T E G L I C H
1
Vinyl-Olefines and Sesquiterpenes in the Root-Oil of Senecio
isatideus W. B O L A N D , L . J A E N I C K E , and A . B R A U N
E R 5
Carotenoid Composition of Chlorophyll-Carotenoid-Proteins from
Radish Chloroplasts H . K . L I C H T E N T H A L E R , U . P R E N
Z E L , and G. K U H N 10
Carotenoid Biogenesis in the Stick Insect, C a r a u s i u s
morosus, during a Larval Instar H . K A Y S E R 13
Hydroxylation of the B-Ring of Flavonoids in the 3'- and
5'-Position with Enzyme Extracts from Flowers of Verbena h y b r i
d a G. S T O T Z and G. F O R K M A N N 19
Dissociation of F A D from The N A D (P) H-Nitrate Reductase
Complex from Ankistrodesmus b r a u n i i and Role of Flavin in
Catalysis M . A . D E L A R O S A , A . J. M Ä R Q U E Z , and J. M
. V E G A 24
Spectrophotometric Isolation of Kinetically Different Pools of
P-700 and Their Correlation to the Re-duction of N A D P by
Isolated Chloroplasts. I. The Effect of Light Quality and Intensity
H . J . R U R A I N S K I , R. G E R H A R D T , and G. M A D E R
31
Chemical Aspects of the Mutagenic Activity of the Ascorbic Acid
Autoxidation System W. H . K A L U S , W. G. F I L B Y , and R. M Ü
N Z N E R 40
Mevinolin: A Highly Specific Inhibitor of Micro-somal
3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase of Radish Plants T.
J . B A C H and H . K . L I C H T E N T H A L E R 46
Free Activation Energies and Activation Volumes f or the Amide
Rotation in Some Peptides Studied by High Pressure ^ - H i g h
Resolution N M R H . H A U E R , H.-D. L Ü D E M A N N , and R. J A
E N I C K E 51
Investigation of Large Intramolecular Movements within
Metmyoglobin by Rayleigh Scattering of Mössbauer Radiation (RSMR) Y
. F . K R U P Y A N S K I I , F . P A R A K , V . I. G O L D A N S
K I I , R. L . M Ö S S B A U E R , E . E . G A U B M A N , H . E N
G E L M A N N , and I. P. S U Z D A L E V 57
On the Free RadicaUnduced Aggregation of Ribo-nuclease — A Pulse
Radiolysis Study Using the Light Scattering Detection Method H . S
E K I and W. S C H N A B E L 63
Dark Oxidation of Unsaturated Lipids by the Pho-toxidized
8-Methoxypsoralen A . Ya . P O T A P E N K O , M . V . M O S H N I
N , A . A . K R A S N O V S K Y J R . , and V . L . S U K H O R U K
O V 70
Growth Specific Thylakoid Differentiation of the Endocyanelle C
y a n o c y t a k o r s c h i k o f f i a n a in the Symbiotic
Association with C y a n o p h o r a p a r a d o x a (In German) P.
B R A N D T , S. M A R T E N , B . M Ü L L E R , and W. WlESSNER
75
Differential Fluorescence and Kinetic Studies on the
Template-Binding of R N A Polymerase from Pars-ley and E s c h e r
i c h i a c o l i K . G R O S S M A N N , H . B I S S W A N G E R ,
and H . U . SEITZ 81
Sensitivity of E s c h e r i c h i a c o l i to Viral Nucleic
Acid. X V I . Temperature Conditions for C a 2 + Dependent D N A
Uptake in E s c h e r i c h i a c o l i A . T A K E T O 87
Studies on 1 80 2-Uptake in the Light by Entire Plants of
Different Tobacco Mutants R. ISHII and G. H . S C H M I D 93
Immobilization of Yeast Gells by Radiation-Induced
Polymerization T. F U J I M U R A and I. K A E T S U 102
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Contents IV
N a n o c h l o r u m e u c a r y o t u m : a Very Reduced
Coccoid Species of Marine Chlorophyceae C. W I L H E L M , G.
EISENBEIS, A . W I L D , and R. Z A H N 1 0 7
Growth and Photosynthesis of N a n o c h l o r u m eu-c a r y o
t u m , a New and Extremely Small Eucaryotic Green Alga C. W I L H
E L M and A . W I L D 1 1 5
Kinetics of Proton-Hydroxyl Transport across Leci-thin Vesicle
Membranes as Measured with a Lipoid pH-Indicator W . G. P O H L 1 2
0
Notes 1 H-NMR- and ESR-Investigations on the Structures
of Dehydroascorbic Acid and the Semidehydro-ascorbate Radical
(In German) H . S A P P E R , A . P L E Y E R - W E B E R , and W .
L O H M A N N 1 2 9
Hydroxyrutacridone Epoxide, a New Acridone A l -kaloid from R u
t a g r a v e o l e n s (In German) U . E I L E R T , B. W O L T E
R S , A . N A H R S T E D T , and V . W R A Y 1 3 2
Flavonol 3-O-Methyltransferase in Plant Tissues V . D E L U C A
, G. B R U N E T , H . K H O U R I , R. I B R A H I M , and G. H R
A Z D I N A 1 3 4
Enzymatic Hydroxylation of Ruthenocen and Osmo-cene (In German)
M . S C H N E I D E R , M . W E N Z E L , and G. S C H A C H S C H
N E I D E R 1 3 6
Neutral Red Fluorescence of Chromatin: Specificity and Binding
Mechanism R. H . E S P E L O S I N and J. C. S T O C K E R T 1 3
9
Proteinase-Inhibitors in Albumin Glands of A c h a t i n a f u l
i c a
R. 0 . O K O T O R E and G. U H L E N B R U C H 1 4 2
Erratum 144
Con ten t s of N u m b e r 3/4
O r i g i n a l C o m m u n i c a t i o n s
Structure Elucidation of Pterosupin from P t e r o -c a r p u s
m a r s u p i u m , the First Naturally Occurring
C-Glycosyl-jß-hydroxy-dihydrochalcone D . A D I N A R A Y A N A , K
. V . S Y A M A S U N D A R , 0 . S E L I G M A N N , and H . W A G
N E R 145
A New Catechin Glycoside from P o l y p o d i u m v u l g a r e
(In German) C. K A R L , G. M Ü L L E R , and P . A . P E D E R S E
N 148
On the Essential Oil Components from Blossoms of A r t e m i s i
a v u l g a r i s L . (In German) K . M I C H A E L I S , 0 . V O S
T R O W S K Y , H . P A U L I N I , R . Z I N T L , and K . K N O B
L O C H 152
Phenazine and Phenoxazinone Biosynthesis in B r e v i b a c t e
r i u m i o d i n u m R . B . H E R B E R T , J . M A N N , and A .
R Ö M E R 159
Separation, Partial Purification and Characteriza-tion of a
Fatty Acid Hydroperoxide Cleaving Enzyme from Apple and Tomato
Fruits P. S C H R E I E R and G. L O R E N Z 165
The Site of Indole-3-acetic Acid Synthesis in Mesophyll Cells of
S p i n a c i a o l e r a c e a B . H E T L M A N N , W . H Ä R T U
N G , and H . G I M M L E R 174
Rubins and Rubinoid Addition Products from Phycocyanin W . K U F
E R and H . S C H E E R 179
Isolation of a 6-Type Cytochrome Oxidase from Membranes of the
Phototrophic Bacterium Ehodopseudomonas c a p s u l a t a H . H Ü D
I G and G. D R E W S 193
Tetrapyrrol Derivatives Shown by Fluorescence Emission and
Excitation Spectroscopy in Cells of Ehodopseudomonas c a p s u l a
t a Adapting to Phototrophic Conditions J . B E C K and G. D R E W
S 199
Formation of Hydrocarbons by Photobleaching Cyanobacterium,
Anacystis n i d u l a n s G. S C H M E T T E R E R 205
Peptidoglycan and Protein, the Major Cell Wall Constituents of
the Obligate Halophilic Bac-terium R h o d o s p i r i l l u m
salexigens M . H . T A D R O S , G. D R E W S , and D . E V E R S
210
CO2 Fixation in A n a b a e n a c y l i n d r i c a G. D Ö H L E
R 213
On the Occurrence of Monoacylglycerol Derivatives in L ip id
Metabolism of Chloroplasts A . S A U E R and K . - P . H E I S E
218
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Contents
Possible Models of Murein and Their Fourier Transforms H . F O R
M A N E K 226
Binding of Antibodies onto the Thylakoid Mem-brane. V I I .
Localization of Coupling Factor of Photophosphorylation in the
Lamellar System of Chloroplasts from A n t i r r h i n u m m a j u
s A . R A D U N Z and R . M E I E R 236
Photophosphorylation by Chloroplasts: Effects of Low
Concentrations of Ammonia and Methyl-amine C. G I E R S C H 242
Digitonin Precipitable Sterols Inducing the Ag-gregation of
Prolamellar Body-Like Structures from Completely Dissolved P L B
Components A . Nis ius , M . M Ü L L E R , and H . G. R U P P E L
251
Isolation of PS II-Particels with i n v i v o Charac-teristics
from E u g l e n a g r a c i l i s , Stamm Z (In German) F . S C H
U L E R , P . B R A N D T and W . W I E S S N E R 256
Absorption and Picosecond Fluorescence Charac-teristics of
Chlorophyll Vesicles as a Function of Temperature S. S. B R O D Y
260
Herbicides which Inhibit Electron Transport or Produce Chlorosis
and Their Effect on Chloro-plast Development in Radish Seedlings.
I. Chloro-phyll a Fluorescence Transients and Photosys-tem II
Activity K . H . G R U M B A C H 268
Use of Isolated Leaf Cells of A b u t i l o n t h e o p h r a s
t i to Localize the Action of Two Aminotriazinone Herbicidal
Derivatives K . K . H A T Z I O S 276
Structure Investigations of Agonists of the Natural
Neurotransmitter Acetylcholine I I [1]. X-ray Structure Analysis of
Trimethyl(4-oxopentyl)-ammonium-chloride A . G I E R E N and M . K
O K K I N I D I S 282
Bile Salt Delipidation, Residual Phospholipids and Reactivation
of the Ca 2 + -ATPase from Sarco-plasmic Reticulum G. S W O B O D A
and W . H A S S E L B A C H 289
V
The Influence of Detergents on the C a 2 + - and M g 2 +
-Dependent Adenosine Triphosphatase of the Sarcoplasmic Reticulum H
. L Ü D I , B . R A U C H , and W . H A S S E L B A C H 299
Inhibition of Acetyl CoA Carboxylase by a High Molecular Weight
Protein in Rat Liver M . N . A B D E L - H A L I M and S. Y . K . Y
O U S U F Z A I 308
Subcellular Fractionation of Bone Marrow-Derived Macrophages:
Localization of Phospholiphase A i and A2 and
AcyI-CoA:l-Acylglycero-3-phos-phorylcholine-O-acyltransferase E . E
. K R Ö N E R , H . F I S C H E R , and E . F E R B E R 314
Periodicities of Dinucleotide Self-Information Val-ues in 0X174
D N A N . B . F U R L O N G and C. F . B E C K N E R 321
Growth Characteristics of i n v i t r o Cultured Ehrlich Ascites
Tumor Cells under Anaerobic Conditions and affcer Rearation
R. M E R Z and F . S C H N E I D E R 326
Notes
: 3-O-Methyl, 8-C-methyl Quercetin a New Flavonoid from D a s y
l i r i o n a c r o t r i c h u m and X a n t h o r r h o e a h a s
t i l i s , Liliales (In German) C. L A R A C I N E , J . F A V R E
- B O N V I N , and P . L E B R E T O N 335
New Flavonoids from the Farina of P i t y r o g r a m m a
Species C. H I T Z , K . M A N N , and E . W O L L E N W E B E R
337
Actinioerythrin in M u l l u s b a r b a t u s L . (Pisces;
Teleostei; Mullidae) (In German) F . -C. C Z Y G A N and A . K R Ü
G E R 340
The Importance of the Position of the Adenosine Linkage in N A D
+ for Its Coenzyme Activity v i a Lactate Dehydrogenase (In German)
E . S C H L I M M E , R . M A T T E R N , and E . SCHOTT 342
Characterization of the Nuclear Lamina in an Insect by
Differential Staining R. M A R X 345
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VI Contents
Trail Orientation of Wood Ants ( F o r m i c a p o l y c t e n a
Förster) (In German) K . H O R S T M A N N 348
Erratum
Conten t s of N u m b e r 5/6
O r i g i n a l Communications
Structure of Echinacoside (In German) H . B E C K E R , W . C H
. H S I E H , R . W Y L D E , C . L A F F I T E , and C . A N D A R
Y
3 5 0
3 5 1
Synthesis of Naturally Occurring Prenylated Naph-thalene
Derivates. Isolation of a New Prenyl-naphthoquinone from G a l i u
m m o l l u g o (In German) L . H E I D E and E . L E I S T N E R 3
5 4
Biosynthesis of Isoflavonoid Phytoalexins: Incor-poration of
Sodium [l,2- 1 3C2]Acetate into Phase-ollin and Kievitone P. M . D
E W I C K , M . J . S T E E L E , R . A . D I X O N , and I. M . W
H I T E H E A D 3 6 3
Diurnal Fluctuation of Quinolizidine Alkaloid Ac-cumulation in
Legume Plants and Photomixotro-phic Cell Suspension Cultures M . W
I N K and T. H A R T M A N N 3 6 9
Volatile Compounds from Cephalic Secretions of Females in two
Cleptoparasite Bee Genera, Epeolus (Hym., Anthophoridae) and
Coelioxys (Hym., Megachilidae) J . T E N G Ö , G. B E R G S T R Ö M
, A . - K . B O R G - K A R L S O N , I . G R O T H , and W . F R A
N C K E 3 7 6
Purification and Properties of Glucosaminephos-phate Isomerase
of Proteus m i r a b i l i s B . C I F U E N T E S and C. V I C E N
T E 381
Effects of Trypan Blue and Related Compounds on Production and
Activi ty of Streptolysin S Y . T A K E T O and A . T A K E T O 3 8
5
Antiviral Activi ty of 2-(a-Hydroxybenzyl)-benz-imidazole and
other 2-Substituted Benzimid-azoles against T M V in Tomato Leaf
Discs A . C. C A S S E L L S and F . M . C O C K E R 3 9 0
Proton NMR-Relaxation Dispersion in Meconium Solutions and
Healthy Amniotic F lu id : Possible Applications to Medical
Diagnosis G. J . B E N E , B . B O R C A B D , V . G R A F , E . H
I L T -B R A N D , P . M A G N I N , and F . N O A C K 4 9 3
Monospecific Antibody against 5-Methyl-Cytidine for the
Structural Analysis of Nucleic Acids S. K . M A S T R O N I C O L I
S , V . M . K A P O U L A S , and H . K R Ö G E R 3 9 9
On the Oxidative Main-Chain Scission of Nucleic Acids — Pulse
Radiolysis Studies (In German) 0 . D E N K and W . S C H N A B E L
4 0 5
Binding Constants, Binding Enthalpies and E n -tropies of the
Non-Competitive and the Com-petitive Binding of Acriflavine,
Tetramethylacri-flavine and Acridine Orange to D N A (In German) H
. W I L L E , J . P A U L U H N , and H . W . Z I M M E R -mANN 4 1
3
Possible Orientation of the Fatty Acid Chains in
Lipopolysaccharide H . F O R M A N E K 4 2 8
Screening for Highly Active Plasmid Promoters via Fusion to
ß-Galactosidase Gene A . R O S N E R , M . G O R E C K I , and H .
A v i v 4 4 1
Anomalous Reduction of Cytochrome b in Highly Purified Complex I
I I from Baker's Yeast F . F . D E L A R O S A and G. P A L M E R 4
4 5
Changes in the Fluorescence Emission Spectrum of C h l o r e l l
a emersonii Induced by Cold Treatment; a Possible Regulative
Feature of Energy Uptake G. H A R N I S C H F E G E R and H . J A R
R Y 4 4 8
Studies of Photosynthesis Inhibition by Phytolu-minography R . B
L A I C H , 0 . B A C H M A N N , and I. B A U M -B E R G E R 4 5
2
A Study of State Changes in Chlorella: The Effect of Un coupler
and Energy Transfer Inhibitors P. V . S A N E , D . F U R T A D O ,
T. S. D E S A I , and V . G. T A T A K E 4 5 8
Adaptation of Chloroplast-Ultrastructure and of
Chlorophyll-Protein Levels to High-Light and Low-Light Growth
Conditions H . K . L I C H T E N T H A L E R , G. K U H N , U . P R
E N Z E L , C. B U S C H M A N N , and D . M E I E R 4 6 4
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Contents VII
Protochlorophyllide Photoeonversion Initiates the Transformation
of Reaggregated Prolamellar Body Tubules i n v i t r o M . M Ü L L
E R and A . N I S I U S 476
The Sarcoplasmic Reticulum of Smooth Muscle Fibers L . R A E Y M
A E K E R S 481
Histochemical Demonstration of an ATP-Dependent Ca 2 + -Pump in
Bullfrog Myocardial Cells R . M E Y E R , W . S T O C K E M , M . S
C H M I T Z , and H . G. H A A S 489
Ca Entry and Contraction as Studied in Isolated Bovine
Ventricular Myocytes G. I S E N B E R G 502
Fluorescence Energy Transfer between ATPase Monomers in
Sarcoplasmic Reticulum Recon-stituted Vesicles, in the Presence of
Low Con-centrations of a Nonionic Detergent P . C H A M P E I L , J
. - L . R I G A U D , and M . P . G I N -G O L D 513
Functional Significance of Quaternary Organization of the
Sarcoplasmic Reticulum Ca 2 + -ATPase J . V . M 0 L L E R , T. S. M
A H R O U S , J . P . A N D E R S E N , and M . L E M A I R E
517
Equilibrium Constants for Some Steps of the Reac-tion Cycle of
the Sarcoplasmic Reticulum Calcium Pump C. T A N F O R D and D . W
. M A R T I N 522
Phosphorylation of Ca 2 + -ATPase by Inorganic Phosphate in
Water-Organic Solvent Media: D i -electric Constant and Solvent
Hydrophobicity Contribution A . D E SOTJZA O T E R O and L . D E M
E I S 527
ATPases of the Cat Carotid Body and of the Neigh-bouring
Ganglia
H . S T A R L I N G E R 532
Notes Synthesis of 6-Chlorogenistein
A . L E V A I and A . L . T Ö K E S 5 4 0
Effect of Penicillins on the Level of Inorganic Pyrophosphatase
in E s c h e r i c h i a c o l i K 12 E . I. K U K K O and J . K .
H E I N O N E N 542
Hyperglycemia in the Fresh Water Field Crab ( O z i o t e l p h
u s a senex senex) Produced by a Pesti-cide (BHC) P . S R E E N I V
A S U L A R E D D Y , S. B . R A M E S H B A B U , and R . R A M A
M U R T H I 5 4 5
Coenzyme Binding at Different Ionization States of Cytoplasmic
and Mitochondrial Malate Dehydro-genase K . S C H W E R D T F E G E
R , C. W O E N C K H A U S , D . M . P A R K E R , and J . J . H O
L B R O O K 5 4 7
A New Method to Prepare Membrane Fractions Containing
Ionophore-Stimulated ATPase from Pumpkin Hypocotyls ( C u c u r b i
t a m a x i m a , L . ) G. F . E . S C H E R E R 5 5 0
Conten t s of N u m b e r 7/8
O r i g i n a l C o m m u n i c a t i o n s
E S R Studies on Platinum Uracil Blue H . N E U B A C H E R , M
. S E U L , and W . L O H M A N N 5 5 3
Roseanolone: A New Diterpene from Hypoestes rosea
J . I. O K O G U N , A . A . A D E S O M O J U , G. A . A D E S
I D A , H . J . L I N D N E R , and G. H A B E R M E H L 5 5 8
6-C-a-L-Rhamnopyranosylapigenin 7-0-/?-D-gluco-pyranoside
(Isofurcatain 7-0-/?-D-glucoside), a New Flavone Glycoside from M e
t z g e r i a f u r c a t a K . R . M A R K H A M , R . T H E O D O
R , R . M U E S , and H . D . Z I N S M E I S T E R 5 6 2
On the Essential Oil Components from K u m u l u s l u p u l u s
L . var. neomexicanus Nels. & Cockerell. I. Contribution K . K
N O B L O C H , H . P A U L I N I , C. E L E Y , J . H . E L E Y ,
E . Z I E G L E R , H . B R A N D A U E R , K . M I C H A E L I S ,
and O. V O S T R O W S K Y 5 6 5
Demethylation, Methylation and 3'-Hydroxylation of Isoflavones
by F u s a r i u m Fungi K . - M . W E L T R I N G , K . M A C K E
N B R O C K , and W . B A R Z 5 7 0
Accumulation of Acridone-Epoxides in Callus Cul-tures of R u t a
graveolens Increased by Coculture with Not Host-Specific Fungi (In
German) B . W O L T E R S and U . E L L E R T 5 7 5
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VIII Contents
Precursors of Cyclic Polysulphides in Seeds of P a r -k i a
speciosa Hassk. (In German) R. S U S I L O and R. G M E L I N
584
Formation of Flavonol 3-O-Diglycosides and Fla-vonol
3-O-Triglycosides by Enzyme Extracts from Anthers of T u l i p a
cv. Apeldoorn G . K L E I N E H O L L E N H O R S T , H . B E H R E
N S , G . P E G E L S , N . SRTTNK, and R . W I E R M A N N 587
Polynucleotide Phosphorylase from a Cyanobac-terium
(Synechococcus s p . ) : Subunit Composition and Properties W . - T
. N O L D E N and G . R I C H T E R 600
DEAE-Dextran Induced Increase of Membrane Permeability and
Inhibition of Photosynthesis in D u n a l i e l l a p a r v a H . G
I M M L E R and G . L O T T E R 609
High- and Low-Affinity Binding of Photosystem II Herbicides to
Isolated Thylakoid Membranes and Intact Algal Cells H . L A A S C H
, K . P F I S T E R , and W . U R B A C H 620
Herbicides which Inhibit Electron Transport or Produce Chlorosis
and Their Effect on Chloro-plast Development in Radish Seedlings.
II. Pig-ment Excitation, Chlorophyll Fluorescence and
Pigment-Protein Complexes C. B Ü S C H M A N N and K . H . G R U M
B A C H 632
Herbicides which Inhibit Electron Transport or Produce Chlorosis
and Their Effect on Chloro-plast Development in Radish Seedlings.
III . Plastid Pigment and Quinone Composition K . H . G R U M B A C
H 642
Effects of Substituted
2-Phenylamino-l,4,5,6-tetra-hydropyrimidines on A T P Formation in
Isolated Spinach Chloroplasts G . V A N D E N B E R G , M . B R A N
D S E , and J . T I P K E R 651
A Cyanobacterial ATPase Distinct from the Cou-pling Factor of
Photophosphorylation W . L O C K A U and S. P F E F F E R 658
The Anatomy of the Sarcoplasmic Reticulum in Vertebrate Skeletal
Muscle: Its Implications for Excitation Contraction Coupling J . R.
S O M M E R , M . D . 665
Sodium-Potassium Movement and the Regulation of Cardiac Muscle
Activi ty J . D A U T and R. R U D E L 679
Regulation of the Sarcoplasmic Reticular C a 2 +
Transport ATPase by Phosphorylation and Dephosphorylation L . M
. G . H E I L M E Y E R , Jr . and M . V A R S A N Y I 682
Equilibrium and Kinetic Studies of Calcium Trans-port and ATPase
Activi ty in Sarcoplasmic Re-ticulum G . I N E S I , M . K U R Z M
A C K , D . K O S K - K O S I C K A , D . L E W I S , H . S C O F A
N O , and H . G U I M A R A E S -M O T T A 685
Comparative Studies on the ATP-Bindung Sites in Ca2+-ATPase and
(Na+ + K+)-ATPase by the Use of ATP-Analogues W . S C H O N E R , E
. H . S E R P E R S U , H . P A U L S , R . P A T Z E L T - W E N C
Z L E R , H . K R E I C K M A N N , and G . R E M P E T E R S
692
Electromechanical Coupling I. Introduction H . C H . L Ü T T G A
U 706
Electromechanical Coupling I L The Effect of Per-chlorate Upon
Excitation-Contraction Coupling in Frog Skeletal Muscle Fibres M .
G O M O L L A , G. G O T T S C H A L K , and H . - C H . L Ü T T -G
A U 707
Electromechanical Coupling III . Estimation of the Ca Storage
Capacity of the SR by Analysing the Time Course of Caffeine-Induced
Tension Tran-sients of Skinned Muscle Fibres R . T H I E L E C Z E
K 709
Intracellular Membranes as Boundaries for Ionic Distribution. I
n S i t u Elemental Distribution in Guinea Pig Heart Muscle in
Different Defined Electro-Mechanical Coupling States M . F . W E N
D T - G A L L I T E L L I , R . J A C O B , and H . W O L B U R G
712
Notes
New Flavonol Acetates from the Frond Exudate of the Fern N o i h
o l a e n a a s c h e n b o r n i a n a M . J A Y , M.-R. V I R I C
E L , J . F A V R E - B O N V I N , B . V O I R I N , and E . W O L
L E N W E B E R 721
-
Contents
Isolation and Identification of Lathycarpin, a New Pterocarpan
Phytoalexin from L a t h y r u s sativus J . L . I N G H A M and K
. R . M A R K H A M 724
Irregularities in the Circular Dichroism of
Oligo-ribonucleotides W . Z A C H A R I A S and H . F O L L M A N N
727
Adult T-Cell Leukemia-Associated Antigen ( A T L A ) : Detection
of a Glycoprotein in Cell- and Virus-Free Supernatant N . Y A M A M
O T O , J . S C H N E I D E R , Y . H I N U M A , and G. H T J N S
M A N N 731
Conten t s of N u m b e r 9
O r i g i n a l C o m m u n i c a t i o n s
Nonlinear Polarisationoscillations in a Biophysical Model.
System I I : External Dynamics Z . S Z A B O and F . K A I S E R
733
The Phytochemistry of South American N e p h r o m a -Species
(In German) B . R E N N E R , A . H E N S S E N , and E . G E R S T
N E R 739
Detection of Glyceollin on the Cellular Level in In-fected
Soybean by Laser Microprobe Mass Analysis P. M O E S T A , U . S E
Y D E L , B . L I N D N E R , and H . G R I S E B A C H 748
Oxygen-Isotope Effect in Enzymatic Cleavage Re-action of
13-L-Hydroperoxylinoleic Acid to Hexa-nal and
ll-Formyl-d.s-9-undecenoic Acid A . H A T A N A K A , T. K A J I W
A R A , J . S E K I Y A , and T. F U K U M O T O 752
On the Biosynthesis of C30 Carotenoic Acid Gluco-syl Esters in
Pseudomonas r h o d o s . Analysis of c a r -Mutants H . K L E I N
I G and R. S C H M I T T 758
Biosynthesis of Photosynthates and Taxonomy of Algae B . P. K R
E M E R and G. 0 . K I R S T 761
Specific Localization of ß-D-Glucoside Conjugates of
2,4-Dichlorophenoxyacetic Acid in Soybean Vacuoles R . S C H M I T
T and H . S A N D E R M A N N , Jr . 772
IX
Synthesis of Galloyl-Coenzyme A Thioester G. G. GROSS 778
Nitrite Inhibition of Bacterial Dinitrogen Fixation F . C A S T
L L L O and J . C A R D E N A S 784
Energy Transfer Inhibition Induced by Nitrofen B . H U C H Z E R
M E Y E R 787
Natural Inhibitors of Germination and Growth, I. Development of
a Quantitative Biotest and Ap-plication upon Extracts from Husks of
Avena s a t i v a L . (In German) R. K A R L and W . R Ü D I G E R
793
Natural Inhibitors of Germination and Growth I I . Isolation and
Structure of Inhibitors from Avena s a t i v a L . (In German) E .
L O H A U S , I. B L O S , W . S C H Ä F E R , and W . R Ü D I G E
R 802
5-Methylcytosine Content in Erythroleucemic Cells of Rats after
Induction with Dimethylsulfoxid (In German) R . B A U R , N . K L U
G E , H . K R Ö G E R , and H . W O H L E R T 812
Catabolic Properties of 5',5"-Linked Dinucleoside-phosphates in
Rat Liver Nuclei (In German) S. B O R N E M A N N and E . S C H L I
M M E 818
The Interaction of Calf Thymus D N A with Mercuric Acetate and
3,6-Bis-(acetatomercurimethyl)-dioxane. Small-Angle X - R a y
Scattering and Viscosity Studies P. Z I P P E R , G. R I B I T S C
H , J . S C H U R Z , and H . B Ü N E M A N N 824
Growth Characteristics of Anaerobically Treated Ehrlich Ascites
Tumor Cells after Reaeration as Studied by Combination of Flow
Cytometry and Centrifugal Elutriation R. M E R Z and F . S C H N E
I D E R 833
Inverse Relationships between Ecdysteroid Titres and Total Body
Metabolism in Insects K . S L A M A 839
Notes
Yeast Aminopeptidase I I : Rapid Purification Using Affinity
Chromatography of the Periplasmic En-zyme J . K N Ü V E R and K . -
H . R Ö H M 845
-
X Contents
Conten t s of N u m b e r 10
O r i g i n a l C o m m u n i c a t i o n s
On the Biogenesis of Aroma Compounds in Plants and Fruits, X V I
I . Anabolie Properties of Straw-berry Fruit Tissue for the
Biosynthesis of Aroma Compounds (In German) F . D R A W E R T and
R. G . B E R G E R 849
Indole Alkaloids from Cell Suspension Cultures of Stemmadenia t
o m e n t o s a and V o a c a n g a a f r i c a n a J . STÖCKIGT, K
. - H . P A W E L K A , A . R O T H E R , and B . D E U S 857
Degradation of the Isoflavone Biochanin A by F u s a r i u m j a
v a n i c u m U . W I L L E K E and W . B A R Z 861
Changes of C-Phycocyanin in Synechococcus 6301 in Relation to
Growth on various Sulfur Compounds A . S C H M I D T , I. E R D L E
, and H. -P . K Ö S T 870
State of Iron in the Archaebacterium M e t h a n o s a r -c i n
a b a r k e r i Grown on Different Carbon Sources as Studied by
Mössbauer Spectroscopy P. S C H E R E R and C H . SATTER 877
Effect of Aging on the Fluorescence Lifetime of Chloroplasts S.
S. B R O D Y 881
On the Role of Hydrogen Peroxide in Peroxidase Catalyzed
Metabolism of Indole-3-acetic Acid H . J . G R A M B O W 884
Distribution and Effects of Bentazon in Crop Plants and Weeds H
. K . L I C H T E N T H A L E R , D . M E I E R , G . R E T Z L A F
F , and R. H A M M 889
Mechanism of Bleaching in Leaves Treated with Chlorosis-Inducing
Herbicides J . F E I E R A B E N D , T H . W I N K E L H Ü S E N E
R , P . K E M M E R I C H , and U . S C H U L Z 898
Rotating-Field-Induced Rotation and Measurement of the Membrane
Capacitance of Single Mesophyll Cells of A v e n a s a t i v a W .
M . A R N O L D and U . Z I M M E R M A N N 908
The Triton X-100 and High Salt Resistant Residue of
Saccharomyces c e r e v i s i a e Nuclear Membranes K . M A N N and
D . M E C K E 916
Malonate and Krebs Cycle Intermediates Utilization in the
Presence of other Carbon Sources by B h i z o -b i u m j a p o n i
c u m and Soybean Bacteroids D . W E R N E R , W . D I T T R I C H
, and H . T H I E R -F E L D E R 921
Alanine Dehydrogenase from Bacteroids and Free Living Cells of B
h i z o b i u m j a p o n i c u m P. M Ü L L E R and D . W E R N E
R 927
The Tautomerism of Cytosine and Hydroxycyto-sine. A
Quantum-Mechanical Study J . S. K W I A T K O W S K I , B . L E S Y
N G , M . H . P A L M E R , and W. S A E N G E R 937
The Reversibility of the Vitamin C Redox System: Electrochemical
Reasons and Biological Aspects H . S A P P E R , S.-O. K A N G , H
. - H . P A U L , and W. L O H M A N N 942
Coupled Gas Chromatography — Single Cell Re-cording: a New
Technique for Use in the Analysis of Insect Pheromones L . J . W A
D H A M S 947
Evaluation of (Z)-5-Decen-l~ol as an Attractant for Male Larch
Casebearer Moths, C o l e o p h o r a l a r i c e l l a E . P R I E
S N E R , W . A L T E N K I R C H , W. B A L T E N S -W E I L E R ,
and H . B O G E N S C H Ü T Z 953
A Steroid-Binding Protein from Insect Haemolymph Isolated by
Photoaffinity Labelling and Immuno-adsorption L . R E U M , G. K Ä
U S E R , U . E N D E R L E , and J . K o O L M A N N 967
Structure Investigations of Agonists of the Natural
Neurotransmitter Acetylcholine, I I I [1]. X - R a y Structure
Analysis of (2-Ethoxyethyl)trimethyl-ammonium Chloride A . G I E R
E N and M . K O K K I N I D I S 977
Polycation-Bacterium Interactions and Wal l Sub-units as
Endocytosis Factors. Topoisomeraselike Action of Basic Polypeptides
Suggesting a 7th Class of Enzymes: The Stereases S. A N T O H I
985
Tl + -Ions: Influence on Cardiac Contractility R. Z I S K O V E
N , C. A C H E N B A C H , J . W I E M E R , and U . W I N T E R
995
-
Contents
Tl+-Ions: Effects on the Automaticity of Sinoatrial Tissue and
on d F / d £ m a x and ^K2 of Cardiac Pur-kinje Fibres C. A C H E N
B A C H , J . W I E M E R , R . Z I S K O V E N , and U . W I N T E
R 1006
Tl + -Ions: Comparison of the Effects on the Slow Inward Current
and Contractility of Ventricular Tissues J . W I E M E R , R . Z I
S K O V E N , and C . A C H E N -B A C H 1015
Differentiation in V o l v o x Carter'i: Study of Pattern
Variation of Reproductive Cells R. G I L L E S and L . J A E N I C
K E 1023
Thermodynamic Approach to a Possible Theory of the Evolution of
a Genetic Code M . F . M A C C H I A T O and A . T R A M O N T A N
O 1031
Notes
B-Decachloro-o-carborane Derivatives Suitable for the
Preparation of Boron-Labeled Biological Macromolecules D. G A B E L
and R. W A L C S Z Y N A 1038
Temperature-Dependent Colour Change in Larvae of the Green
Spider M i c r o m a t a r o s e a (Sparassidae) (In German) A . H
O L L 1040
"Memory" of First Interaction with Physiological or Biologically
Active Foreign Molecules (Benz-pyrene, Gibberelline) in a
Unicellular ( T e t r a -hymena) Model System G. C S A B A , G. N E
M E T H , and P . V A R G H A 1042
On the Nerve Growth Factor (NGF) from Human Glandula
submandibularis (In German) R . R I E M S C H N E I D E R , F . D E
M B E L E , and I. A . K . G H O U R I 1045
Spatio-Temporal Visual Receptive Fields as Revealed by
Spatio-Temporal Random Noise E . H I D A and K E N - I C H I N A K
A 1048
On the Taxonomic Position of Hypocomidae (Ciliata) (In German) K
. G. G R E L L and A . M E I S T E R 1050
XI
Conten t s of N u m b e r 11/12
O r i g i n a l C o m m u n i c a t i o n s
Composition of Epicuticular Waxes from Fruits of J o j o b a ( S
i m m o n d s i a chinensis [Link] Schneider) P . - G . G Ü L Z
1053
Biliverdin I X a, Intermediate and E n d Product of Tetrapyrrole
Biosynthesis H.-P . K Ö S T and E . B E N E D I K T 1057
The Oxidative Degradation of L-Ascorbic Acid via an
a-Ketoaldehyde S.-O. K A N G , H . S A P P E R , and W . L O H M A
N N 1064
Further Observations on the Source of Nitrogen in Phenazine
Biosynthesis A . R Ö M E R and R . B . H E R B E R T 1070
Photosynthetic Adaptation in Synechococcus Cells G . D Ö H L E R
and J.-C. L E C L E R C 1075
Interrelationship between Quinolizidine Alkaloid Producing
Legumes and Infesting Insects: E x -ploitation of the
Alkaloid-Containing Phloem Sap of C y t i s u s s c o p a r i u s
by the Broom Aphid A p h i s c y t i s o r u m M . W I N K , T. H A
R T M A N N , L . W I T T E and J . R H E I N H E I M E R 1081
Biological Activities of Sesquiterpene Lactones from H e l i a n
i h u s annuus: Antimicrobial and Cytotoxic Properties; Influence
on D N A , R N A , and Protein Synthesis O. S P R I N G , J . K T J
P K A , B . M A I E R , and A . H A G E R 1087
Bleaching Activity of New 2-Phenylpyridazinones:
Structure-Activity Relationship G . S A N D M A N N and P . B Ö G E
R 1092
Some Observations on the Saponin Accumulation in Oat Seedlings
and on the Transformation of the Avenacosides to the Antibiotic
26-Desgluco-avenacosides I L L A U D E N B A C H and J . K E S S E
L M E I E R 1095
Naramycin B , an Antibiotic from Streptomyces griseus Strain 587
with Herbicidal Properties-Fermentation, Isolation, and
Identification D . B E R G , M . S C H E D E L , R . R . S C H M I
D T K . D I T G E N S and H . W E Y L A N D 1100
-
Contents XII
The Intracellular Distribution of Enzymes of the Glycerol Cycle
in the Unicellular Alga D u n a l i e l l a p a r v a H . G I M M L
E R and G. L O T T E R 1107
Osmoregulation in D u n a l i e l l a . Intracellular
Distri-bution of Enzymes of Glycerol Metabolism A . D . B R O W N ,
R . McC. L I L L E Y , and T. M A R E N G O 1115
Multicomponent Mandibular Gland Secretions in Three Species of
Andrena Bees (Hym., Apoidea) G. B E R G S T R Ö M , J . T E N G Ö ,
W . R E I T H , and W . F R A N C K E 1124
The Sex Pheromone of the Silver Y Moth C h r y s o -d e i x i s
e r i o s o m a (Doubleday) in New Zealand M . H . B E N N , R . A
. G A L B R E A T H , V . A . H O L T , H . Y O U N G , G. D O W N
, and E . P R I E S N E R 1130
Structure-Activity-Relation of Analogues and Ho-mologues of
Blepharismone, the Low-Molecular Conjugation Hormone of B l e p h a
r i s m a j a p o n i c u m (In German) M . E N T Z E R O T H and L
. J A E N I C K E 1136
Conformational Analysis of Benzyloxycarbonyl-Protected Peptides
I. K N A C K 1141
Immunochemical Characterization of P o r p h y r i d i u m c r u
e n t u m B-Phycoerythrin: Proof of Cross-Reac-tion between
Chromophore-Free Apoprotein and Holoprotein-Specific Antibodies E .
S E P P , G. W A N N E R , J . E D E R , and H. -P . K Ö S T
1146
X - R a y Studies on Phospholipid Bilayers. II . Poly-morphie
Forms of Dipalmitoyl Phosphatidyl-ethanolamine M . S U W A L S K Y
and E . K N I G H T 1157
Statistical Description of Isotope Exchange Pro-cesses: A
Multisite Model for the 1 8 0 Exchange P. R Ö S C H 1161
Fluorescence Studies on N-(3-Pyrene)Maleinimide-Labeled
Sarcoplasmic Reticulum ATPase in Na-tive and Solubilized Membranes
H . L Ü D I and W . H A S S E L B A C H 1170
Localization of C a 2 + at the Plasma Membrane of Bullfrog
Myocardial Cells R. Meyer, M . SCHMITZ, W . S T O C K E M , and H .
G. H A A S 1180
Release of K + and H + from Poly U in Aqueous Solution upon y
and Electron Irradiation. Rate of Strand Break Formation in Poly U
E . B O T H E and D . S C H U L T E - F R O H L I N D E 1191
Model Reactions for the Degradation of D N A - 4 ' Radicals in
Aqueous Solution. Fast Hydrolysis of a Alkoxyalkyl Radicals with a
Leaving Group in ß-Position Foliowed by Radical Rearrange-ment and
Elimination Reactions G. B E H R E N S , G. K O L T Z E N B T X R G
, and D . S C H U L T E - F R O H L I N D E 1205
Induction of Single- and Double-Strand Breaks in Linear and
Superhelical D N A by Phleomycin (In German) F . S C H M Ü L L I N
G and W. K Ö H N L E I N 1228
Effect of Water Soluble Polymer, Polyethylene-glycol, and
Glass-Forming Compounds on Cell Fusion F . Y O S H I I and I. K A E
T S U 1234
System Analysis of the Circadian Rhythm o i E u g l e n a g r a
c i l i s , I. Linearities and Non-Linearities in the Response to
Temperature Signals W . L O R K , T. K R E U E L S , W. M A R T I N
, and K . B R I N K M A N N 1240
Exogenous and Endogenous Control of Swimming Activi ty in A s t
y a n a x mexicanus (Characidae, Pisces) by Direct Light Response
and by a Circadian Oscillator. I. Analyses of the Time-Control
Systems of an Epigean River Population W. E R C K E N S and W. M A
R T I N 1253
Exogenous and Endogenous Control of Swimming Activi ty in A s t
y a n a x mexicanus (Characidae, Pisces) by Direct Light Response
and by a Circadian Oscillator. II . Features of Time-Con trolled
Behaviour of a Cave Population and their Comparison to a Epigean
Ancestral Form W. E R C K E N S and W . M A R T I N 1266
Functional Regeneration of the Visual System in Teleosts.
Comparative Investigations after Optic Nerve Crush and Damage of
the Retina R. K Ä S T N E R and H . W O L B U R G 1274
Notes
Monoterpenes from the True Bug H a r p o c e r a t h o r a -c i
c a (Hemiptera) H . -P. H A N S S E N and J . J A C O B 1281
-
Contents
Diterpenes of C h e i l a n t h e s a r g e n t e a , a Fern
from Asia E . W O L L E N W E B E R , P . RTTEDI,
and D . S. S E I G L E R 1283
Composition of Phospholipids in Seed Oil of J o j o b a ( S i m
m o n d s i a c h i n e n s i s [Link], Schneider) P. G . G Ü L Z
and C. E I C H 1286
Adenine Phosphoribosyltransferase Activity in Mitochondria of C
a t h a r a n t h u s roseus Cells F . H I R O S E and E L A S H I
H A R A 1288
Inactivation of the Sarcoplasmic Reticulum
Cal-cium-Transport-ATPase by Lasolocid in Com-bination with Triton
X-100 W . H A S S E L B A C H , H . L Ü D I , and A . M I G A L A
1290
Small- and Large-Angle X - R a y Scattering Studies of Counter
Ion Influence on t R N A Conformation P. W I L H E L M , I. P I L Z
, G . D E G O V I C S , and F . V O N D E R H A A R 1293
XIII
Isolation and Synthesis of Hemoregulatory Peptide W . R .
Paukovits and 0 . D . Laerum 1297
Erratum
To N . Yamamoto, J . Schneider, Y . Hinuma, and G . Hunsmann
(37c, 731-732 [1982]) 1300
Subject Index 1301
Authors Index 1327
Errata in this volume
To A . Hager and M . Heimle (36 c, 997 [1981]) 144
To J . Kesselmeier and D . Strack (36c, 1072 [1981]) 350
To N . Yamamoto, J . Schneider, Y . Hinuma, and G . Hunsmann
(37c, 731 [1982]) 1300
-
Rubins and Rubinoid Addition Products from Phycocyanin * W.
Kufer and H . Scheer
Botanisches Insitut der Universität München, Menzinger Straße
67, D-8000 München 19
Z. Naturforsch. 37c, 179-192 (1982); received December
15,1981
Phycocyanin, Phycorubin, Bile Pigments, Biliverdin, Bilirubin
The verdin-type chromophore of denatured C-phycocyanin (1) from
Spirulina platensis is
reduced to the corresponding rubin (2 a) by sodium borohydride.
The structure assigned is in agreement with the uv-vis
spectroscopic properties of the product and was deduced from model
studies with free bile pigments.
Analogous model studies using sodium dithionite demonstrated a
two-fold reactivity for this reagent, leading to products which are
both of the rubin spectral type under the conditions tested. True
rubins (10,22-dihydrobilindions) are formed in low yield only if an
excess of reagent is used in methanol/water mixtures. It is
accompanied by polar addition product(s) of the same spectral type,
which are generally formed exclusively. In particular, no bili
rubin was formed under the reaction conditions previously applied
for the chemical modification of phycobiliproteins and phytochrome.
From this finding and from the strikingly different properties of
the borohydride and dithionite products, of phycocyanin upon
renaturation, the dithionite product is suggested to be a rubinoid
addition product (2 b) rather than a hydrogenation product.
In contrast to the dithionite addition product 2 b of
phycocyanin, the chromophore of the true phycorubin (2 a) remains
stable upon renaturation. The uv-vis spectral properties of the
chromophore are not markedly different whether the apoprotein is in
its native or denatured State. The different electrophoretic
mobilities of native (renatured) phycocyanin compared to the
renatured borohydride product suggest that these two have different
protein conformations.
The preparation of these phycorubins renders the extensive
techniques of bilirubin chemistry applicable in the study of
biliproteins.
Introduction
Bile pigments, open-chain tetrapyrrolic Com-pounds, serve
important functions in plants. In contrast to mammalian bile
pigments derived from heme degradation, they are biosynthesized as
pho-toreceptors which are covalently bound to apopro-teins, thus
forming "biliproteins" (for a recent review, see [1]). Two
functionally different groups can be distinguished: Firstly the
phycobiliproteins as light harvesting pigments of photosynthesis in
cyanobacteria, red and cryptophytan algae, and secondly phytochrome
and the phycochromes as photomorphogenetic receptor pigments of
higher plants and some algae.
The physico-chemical properties of the bile pig-ment
chromophores are profoundly influenced by the native proteins. This
is mainly due to non-covalent protein chromophore interactions
which are essential in biliproteins to optimize the proper-
Abbreviations: PC, phycocyanin; tlc, thin layer chromato-graphy;
mob, electrophoretic mobility relative to Standard. * Studies on
Plant Bile Pigments 12; part 11 of this series: W. Kufer and H.
Scheer, Angew. Chem., in press. Reprint requests to Prof. Dr. H.
Scheer. 0341-0382/82/0300-0179 $01.30/0
2a R = H 2c R = SO; 2b R = SOr 2d R = S - C H 2 - CH 2 OH
-
180 W. Kufer and H. Scheer Rubins and Rubinoid Addition Products
from Phycocyanin
ties of the chromophores in their function as photo-receptors.
This can be easily seen from a com-parison of native and
(reversibly) denatured pig-ments (see ref. [1] for a survey).
Recently, we devel-oped a technique for the selective chemical
modifi-cation of biliprotein chromophores [2, 3]. The ex-periments
reported here have been performed to yield more detailed
information about the structure of the chemically modified
products, and the non-covalent protein chromophore interactions in
bili-proteins. The previous experiments of this type were carried
out with the reducing agents dithionite, sulfite and
2-mercaptoethanol, and pigments of the rubinoid spectral type were
obtained [3]. Here, we wish to report the reaction of C-phycocyanin
(1) (C-PQ and some free bilins as model Compounds with sodium
borohydride. This reagent has been used [4, 5] for selective
reduction of bilindiones ("biliverdin" type chromophores like (3))
to 10,22-dihydrobilindiones ("bilirubin" type chromophores like
(4))*.
Results
R e a c t i o n of p h y c o c y a n i n w i t h s o d i u m b o
r o h y d r i d e
The reactions are summarized in Scheme 1. When PC (concentration
ränge 30-130 U M chromo-phores**) denatured by 8 M urea in 50 mM
sodium phosphate buffer, p H 7.5, was treated at 0 °C with solid
sodium borohydride (25-40 mM***), the bands in the uv-vis spectrum
at 602 nm ( e = 15400 per chromophore) and 355 nm ( e = 37000) [31
dis-appeared completely after 20-30 min and a new band at 416 nm
arose simultaneously. The p H of the Solution increased to about 9,
which could be avoided without significant changes in reactivity by
using a buffered Solution of sodium borohydride. Using PC (48 U M
chromophores) and 13 mM N a B H 4 , a 90% decrease of the 602 nm
band was observed within 30 min. The reaction went to
completion
within additional 30 min after a further addition of the same
amount of borohydride (Fig. 1)*. Assum-ing a 1:1 stoichiometry with
no by-product formed, £ 4 1 6 = 24200 (per chromophore) was
calculated from the spectral changes, and with £ 6 0 2=15400 [3]
for the educt chromophores**. The vis-spectrum remained unaltered
when the reducing agent was removed by gel Filtration on a column
equilibrated with 8 M urea. The yellow colour of the borohydride
product also remained unchanged after simultanous removal of urea
and borohydride by gel filtration (/.max = 418 nm). The extinction
coefficient of the pigment thus obtained was determined by
unfolding the protein again, by addition of solid urea to a final
concentration of 8 M . With £ = 24200 as reference value for the
denatured pigment (see above) and allowing for the dilution caused
by addition of urea, e418 - 21900 was obtained for
"native"phycorubin.
The electrophoretic mobility of the product ob-tained after
removal of urea and borohydride dif-fered from that of native PC in
tris-glycine electro-phoresis. With equal protein concentrations
applied, the yellow product had a higher mobility (mob = 0.72 vs.
bromophenolblue as reference Compound) as compared to PC (mob =
0.59) (Fig. 2). They were identical, however, in SDS-gel
electrophoresis hav-ing a Single band corresponding to M W = 19900
in coelectrophoresis using the System of Weber and Osborn [6] (a
Separation into subunits was generally not obtained with PC from S
p i r u l i n a p l a t e n s i s ) . Reaction of the yellow
pigments with diazotized ethyl anthranilate (5-10 fold molar
excess) yielded uv-vis spectroscopically identical pigments with a
long wavelength band at 485 nm, irrespective of the removal of only
N a B H 4 or both N a B H 4 and urea before the reaction.
Addition of solid sodium borohydride (43 mM) to native PC (6.6 U
M chromophores, calculated from £ 6 2 0=98700/chromophore [3]) led
to only partial con-version to the yellow pigment. After 10 min
reaction
* See ref. [43] for the nomenclature of bile pigments, and
formulas for the numbering System. In addition, the non-systematic
terms "rubin" and "verdin" are used to characterize pigments
bearing the conjugation Systems of bilirubin and biliverdin,
respectivley.
** Concentrations throughout are given for chromo-phores. Since
monomeric (
-
W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products
from Phycocyanin • ca. U OmM Na B H 6
P C n a t i v e • "Phycocyanorub»n M n a t i v e
181
(A)
\ « « « = 620nm A. mox 3 type A chromophores
\ =620, 418 nm * max ' type A • type D chromophores
• 8M urea
' Phycorubin" r e n a t u r e d
Xmax = i , 8 n m
3type D chromophores
PC denatured • 2U mM NaBH, "Phycorubin" denatured Xmax = 6 0 2 n
m
3type B chromophores
• 5-1fJ- 3M Dithionite
= 418 nm
PC native + \ = 620 nm ^max 3type A chromophores
-D i th ion i te
Amax 3 type D chromophores
"Phycocyanosulfinat"nat jve \ = 620, 418nm
type A +type C chromophores
(B)
• 8M urea
-u rea ; 0 or 5 -10"* M Dithionite
+ 8 M urea
5-10' 3 M Dithionite
-urea j
5-lO" 3 M Dithionite
PC • 5-10~4 M Dithionite
denatured ' P C - Sulfinat'
- Dithionite denatured v max 3 type B chromophores
: L 18nm ^max 3 type C chromophores
Type A TypeB TypeC: R = SO; TypeD: R = H
Scheme 1. Reaction scheme of the reversible renaturation of PC
from Spirulina platensis, and of the reactions with sodium
borohydride (A) and sodium dithionite (B). The chromophore
structures A - D are schematic representations of the chro-mophore
geometry. Structure A is representative of an extended conformation
without major steric hindrance of the /?-pyr-rolic substituents.
Structure B is the cyclic-helical conformation found for biliverdin
in Solution [22, 23]. Structures C and D have been drawn with the
two dipyrromethenone units nearly perpendicular to each other,
similar to the crystal structu-re of bilirubin [46, 47]. The
reactions of PC with sodium sulfite and 2-mercaptoethanol
principally follow Scheme 1B as well but different concentrations
of the reagents are needed.
-
194 H. Hüdig and G. Drews • Isolation of a 6-Type Cytochrome
Oxidase from Rps. capsulata
P u r i f i c a t i o n of c y t o c h r o m e o x i d a s e
A l l Operations were performed at 4 °C unless stat-ed
otherwise. The membrane fraction was re-suspended in TRIPE buffer
at a concentration of about 6 mg protein per ml. Triton X-100 (10%
stock Solution) in TRIPE buffer was added dropweise to the
Suspension under stirring to a final concentration of 1.3% (w/v,
protein to detergent ratio of 1 : 2). Af-ter incubation for 30 min
insoluble material was pre-cipitated at 145,000 xg , 90 min, 4 °C.
The super-natant contained about 90% of the cytochrome oxi-dase.
The enzyme extract was diluted with TRIPE buffer to a final
concentration of 1% Triton X-100.
10 ml of the supernatant (4 mg protein/ml) were applied to a
column (1x8 cm) of D E A E Sepharose CL-6B (Pharmacia Freiburg)
equilibrated with TRIPE buffer plus 1% Triton. The column was
wash-ed with 30 ml of TRIPE buffer plus 1% Triton and eluted with a
linear gradient of 0 - 1 M K C l (2 x 40 ml). Fractions of 3 ml
were collected and test-ed for cytochrome oxidase activity.
The peak fractions eluted at 0.3 M K C l were pool-ed and
desalted as described in [10]. 9 ml of the de-salted enzyme
Solution (5.6 mg protein) was then ap-plied to a cytochrome
c-thiol-activated Sepharose 4B column (1x4 cm; Pharmacia)
equilibrated with TRIPE buffer plus 1% Triton X-100. The coupling
of cytochrome c from Saccharomyces c e r e v i s i a e (Sigma,
München, type VIII) and the preparation of the cys-teine
inactivated column were performed as describ-ed in [12]. The
binding capacity of the column was 7.7 mg cytochrome c per ml of
packed material. The column was washed with 20 ml of TRIPE buffer
and then eluted with a linear gradient of 0-0.5 M K C L Solution (2
x 40 ml).
D e t e r m i n a t i o n of e n z y m a t i c a c t i v i t
y
Cytochrome oxidase was assayed at 30 °C by fol-lowing the
decrease of absorption at 546 mn in an Eppendorf photometer (type
1101 M) using dithion-ite reduced horse heart cytochrome c as
electron donor (6 = 21 x 103 cmVmmol). The reaction mix-ture
contained 50 mM TRIPE buffer (pH 8), 1 mg ferrocytochrome c and
enzyme Solution in a final volume of 1 ml. The reaction was started
by addition of 10 to 100 u] enzyme Solution. When inhibitors were
used, the enzyme preparation was preincubated for 5 min with the
inhibitor. The reaction was then started by addition of reduced
cytochrome c. Horse
heart ferrocytochrome c was prepared as described in [10] with
the modification that the p H of the fer-rocytochrome c Solution
was adjusted to p H 8 with 1 MTris.
N a t i v e P o l y a c r y l a m i d e g e l e l e c t r o p h
o r e s i s (charge- s h i f t - e l e c t r o p h o r e s i s
)
Electrophoresis in 5% Polyacrylamide slab gels containing 0.1%
Triton X-100 was carried out ac-cording to the method of Simons et
a l [13] with the modification that D T T was added to the gel and
the buffers in 1 mM concentration. The samples were preincubated in
the sample buffer for 15 min at 4 °C. Electrophoresis was performed
at 4 °C with 2 mA/gel track. After the run one track was cut into 2
mm slices. The slices were incubated over night with 200 ul of
TRIPE buffer plus 0.1% Triton and tested afterwards for activity in
the enzyme assay. The slices of the second track were extracted for
4 h with 50 ul of sample buffer (4% SDS) at 60 °C. The extracts
were then subjected to SDS-gel electrophor-esis. The third track
was stained with 0.04% Coo-massie brillant blue in 25% isopropanol
and 10% acetic acid (v/v) and destained with 10% acetic acid and
10% methanol.
Sodium dodecylsulfate P o l y a c r y l a m i d e g e l e l e c
t r o p h o r e s i s
Slab-SDS-gel electrophoresis was performed ac-cording to the
method of Laemmli [14] using 11.5-16.5% acrylamide gradient gels of
1 mm thick-ness. The samples were dialyzed for two days,
lyo-philized and resuspended in sample buffer. A l l sam-ples were
preincubated at 60 °C for 30 min or at 100 °C for 10 min,
respectively.
D e t e r m i n a t i o n of p r o t e i n
Protein concentration was determined by the method of Lowry et a
l . [15]. The presence of Triton X-100 was corrected by addition of
0.5% (w/v) sodi-um dodecylsulfate to the alkali Solution of the
Lowry reagent [16]. Bovine serum albumin was used as Standard.
M e a s u r e m e n t of difference a b s o r p t i o n s p e c
t r a
Difference spectra were obtained with a Perkin-Elmer split beam
spectrophotometer model 330 (Überlingen) using 1 cm light-path
cuvettes at room
-
H. Hüdig and G. Drews • Isolation of a 6-Type Cytochrome Oxidase
from Rps. capsulata 195
Table I. Purification of cytochrome oxidase from
Rhodopseudomonas capsulata strain 37 b4. The purification procedure
and the enzyme assay are described under Materials and Methods.
Activity Protein Specific Purification Yield Activity
Activity
[umol/min • ml] [mg/ml] [umol/min mg protj
(= fold) [%]
Membranes 2.3 6.15 0.37 1 100 Triton extract 3.4 3.9 0.87 2.4
148 DEAE-Sepharose eluate; 1.19 0.19 6.7 17 51 peak fraction
Cytochrome c-thiol- 0.63 0.011 59.7 161 29 activated Sepharose
eluate; peak fraction
temperature. 150 JIM potassium ferricyanide was added to the
reference cuvette and 100 U M sodium dithionite to the sample.
Before recording the spec-trum the cuvettes were kept for 2 min in
the dark.
Results and Discussion
S o l u b i l i z a t i o n of c y t o c h r o m e c o x i d a s
e
Ninety seven percent of the enzyme was solubi-lized at a Triton
concentration of 1.3% and a protein to detergent ratio of 1 : 2.
The activity of the enzyme was enhanced about 30-50%. Lowering the
Tris concentration from 50 mM to 20 mM reduced the solubilization
of the enzyme. A n increase of de-tergent concentration to more
than 1.3% resulted in a loss of activity. Addition of K C l up to 1
M stimulated the activity about 10% (in contrast to NaCl) but had
no effect on the solubilization of the cytochrome oxi-dase
activity.
The detergent N,N-dimethyllaurylamineoxide (LDAO) solubilized
the enzyme activity at a concen-tration of 1.3% nearly 100% but the
half life of the activity dropped to 3 - 5 h. A mixture of L D A O
and other detergents or increasing the ionic strength by addition
of K C l or NaCl caused a complete loss of the activity. The
detergents Brij-58, Tween 20, L u -brol-PX and SDS applied at 4 °C
and at room tem-perature solubilized the enzyme only partially or
re-sulted in inactivation of cytochrome oxidase. Sodi-um cholate
solubilized cytochrome oxidase activity up to 90% but with a lower
yield than Triton. In the presence of 1.3% Triton X-100 the
cytochrome oxi-dase had a half-life of 24 h at 4 °C. Freezing of
the solubilized enzyme even in the presence of 50% gly-cerol
destroyed the activity. The crude Triton-extract lost approx. 20%
of activity at 4 °C in 24 h.
P u r i f i c a t i o n
Triton-extract from membranes was applied to a DEAE-Sepharose C
L 6B column. Enzyme activity eluted shortly after a dark red band
at 300 mM K C l (Fig. 1). The specific activity was increased
17-fold (Table I).
The enzyme fraction which eluted from the DEAE-Sepharose column
contained considerable amounts of cytochrome c besides cytochrome
b. The desalted fraction was loaded on a cytochrome c-thiol
activated Sepharose 4B column. The cytochrome c was covalently
linked through its cysteine residue lo-cated close to the
N-terminus [17] thus leaving free the important lysine residues for
binding cy-tochrome c oxidase and reductase [12]. The enzyme was
not bound to the column when cystein was inac-tivated.
A 2 9 0
FRACTION NUMBER
Fig. 1. Ion exchange chromatography on DEAE-Sepharose
Cl-6B-column (1x8 cm; Pharmacia) in presence of 1% Tri-ton X-100
and Standard buffer (0.05 M Tris-HCl pH 8, 0.1 mM PMSF, 0.1 mM
EDTA). • • Cytochrome oxidase-activity; - -O—O— A 2 9 0 ;
KCl-gradient 0 -1 M KCl , 2 x 40 ml; fractions containing 3 ml.
-
184 W. Kufer and H. Scheer • Rubins and Rubinoid Addition
Products from Phycocyanin
Table I (continued).
Compound and reagent Absorptions A m a x [nm]
A m a x after extraction in CHC13
tlc: ÄrvaluesxlOO Absorptions A m a x [nm]
A m a x after extraction in CHC13 a b c d e f g h
Mesobilirubin, 4 b
MixtureofIII,IXand XIII a-isomers of mesobilirubin
(5b,4b,6b)
424,398 shj), 419m)
66-74
68 74 78
60-69
60
31
31
65
Octaethylbilindion, 7 7 + NaBH 4 !) 7 +
2-mercaptoethanoli,s)
646l) 400,420 sh 426
47 11
2,3-Dihydrooctaethyl-bilindion, 8 8 + NaBH,1) 8 +dithionitej) 8
+ 2-mercaptoethanol*» s)
596 *)
400 398c) 410
65
05
The octaethylbilindion 7 and its 2,3-dihydro-deri-vate 8
similarily yielded yellow products upon treat-ment with solid
borohydride in methanol. 7 reacted smoothly at ambient temperature,
8 only at elevated temperatures (50 °C, under nitrogen). The
conjuga-tion System of 8 is identical to that of C-PC.
Using sodium dithionite instead of borohydride, the results
depended critically on the reaction con-ditions. In an experiment
in methanol/water (1:1, v/v) analogous to the one described above,
a yellow product different from 4 a was obtained from 3 a with 1 mM
dithionite. It migrated with an Äp-value of or close to zero on
silica gel-tlc, developed with neutral or acidic solvent Systems
(a, e, f in Table I). On polyamide-tlc with a basic solvent System,
it mi-grated close to the front (g in Table I). No bilirubin (4 a)
could be detected under these conditions. Upon rising the
dithionite concentration to 24 mM, 4 a and its isomers (5, 6) could
be identified as by-products besides the aformentioned polar
product(s). The product mixture was analyzed quantitatively after
reaction on the preparative scale (3a = 10~ 4M, di-thionite = 2 x
10~2 M). After work up, esterification with diazomethane and
preparative tlc, 4 c was isolated in 4% yield and identified by
uv-vis and tlc comparison with authentic bilirubin dimethylester (4
c). The results obtained with mesobiliverdin (3 b) were analogous
(see Table I).
In another series of experiments, biliverdin was treated with
dithionite in the absence of organic solvents. In particular, the
reaction of 3 a (9 x 10~6) was studied in phosphate buffer (50 mM
sodium phosphate, pH 7.5) containing urea (8 M ) , e. g . under
conditions identical with those used for the reaction of PC [3].
With dithionite concentrations ranging from 2xl0~ 4 to 2 X 1 0 " 2
M , no bilirubin could be identified in the yellow product
mixtures. Chro-matography revealed only polar product(s) (RF = 0 in
System a, as compared to 0.85 for 4 a). The same results were
obtained using buffer without urea.
The conformationally restricted pigments phorca-bilin (9) and
isophorcabilin (10) reacted smoothly, too, with dithionite (see
Table II). Treatment of 9 (13 x 10 - 6 M) with sodium dithionite (5
x 10~5 M) in a buffer/methanol mixture led to decrease of the
Table II. UV-vis spectral data of the biliverdin 11, the
ver-dins 9 and 10 with an extended conformation similar to the one
suggested for the chromophores of native PC [25], and of their
reaction products with reducing agents.
Compound A m a x [nm] in methanol
Phorcabilin-dimethylester, 9 555 562 b)
9+NaBH 4 417 9 + dithionite (0.05 mM) 412 b)
Isophorcabilin-dimethylester, 10 606,690 (sh)
614b) 10+NaBH4 417 10 + dithionite (0.5 mM) 435,455 (sh),
405 (sh)b) Biliverdin IX y-dimethylester, 11 645 l l+NaBH 4 408
11 +dithionite5) c) a) See footnote (s), Table I. b) 50 mM sodium
phosphate buffer, pH 7.5/methanol = 1:1 c) Not determined due to
precipitation.
-
W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products
from Phycocyanin 185
60
Ü0
20
700 600 500 400
« X[nm] Fig. 3. Titration of isophorcabilindimethylester (10)
(9.6X10~6M) in a 1:1 mixture of methanol and phosphate buffer (50
mM, pH 7.5) with sodium dithionite. The absorption spectra were
recorded at the following concentrations of dithioni-te curve 1: 0
M, 2: 5xl0~6 M, 3: 5xl0~ 5 M, 4: 5xl0~ 4 M, and have been recorded
each 5 min after the respective additions of the reagent.
long-wavelength band ( A m a x = 562 nm) by 95%, and the
appearance of only one product band ( X M A X = 412 nm). Titration
of 10 with dithionite (Fig. 3) led to 96% bleaching with 5 x 10~4 M
dithionite, again forming a yellow product ( A m a x = 435,
Shoulders at 435 and 405 nm).
In addition to the results described here, some earlier results
[3] of the reaction of model Com-pounds for the PC chromophore with
sodium sulfite and 2-mercaptoethanol have been incorporated in
Table 1. They have already been published, to-gether with
preliminary results obtained with sodium dithionite.
Discussion
The reaction product of denatured PC with sodium borohydride has
an absorption maximum at 416 nm. Spectroscopically similar products
were found with other reducing agents, viz. sodium dithionite,
sodium sulfite and 2-mercaptoethanol [2, 3] (see also Scheme 1).
This suggests for all these products an interruption of the
conjugated System of the PC chromophore (la) at the C-10 position.
For the rings C and D , this leads to the chromophoric System of
mesobilirubin (4 c, / L m a x = 419 nm in phos-phate buffer
containing 8 M urea). A l l four products have similar extinction
coefficients: £ 4 1 6 = 24200 with borohydride, £ 4 1 8 = 25500
with dithionite [3], £ 4 1 8 = 27900 with sulfite and £ 4 1 8 =
23600 with 2-mercaptoethanol. They amount to roughly 50% of the
value reported for mesobilirubin (4 c) (e4Z3 (CHC13) = 54600 [10]);
thus supporting the general structure 2. For rings A and B, the
interruption of
the conjugation at C-10 leads to a vinylpyrrole chromophore not
absorbing in the visible ränge (e.g. 313 nm for a series of
3,4-dihydro-6(l H)-pyr-romethenons [8, 9] closely related to the
ring A , B-fragment of 2 a). The borohydride product has an
absorption in this spectral ränge ( A m a x = 310 nm) which appears
as a Shoulder of the protein band (̂ max = 280 nm), the other
products could not be investigated in this region due to strong
absorption of the reagents.
Although the phycocyanin reaction products with these four
reductants are uv-vis spectroscopically nearly identical, they
differ in their structure con-cerning the residue R in formula 2.
From uv-vis and tlc-data of the reaction products of model
Com-pounds with sulfite, and from the acid reversible product
formation with 2-mercaptoethanol, the structures 2 c and 2d were
suggested [3] for these pigments arising from addition of the
respective reagents. The pronounced reactivity of C-10 in
bilindiones towards nucleophiles had been inferred first from M O
calculations [12]. After circumstantial evidence [3, 13 a, b],
NMR-data of adducts of thioles with bilindiones in agreement with
the proposed structure 2d have been reported [14, 15], and the
thermodynamic data for related equilibria have been obtained
[15].
Reaction of the same model Compounds with sodium borohydride led
via addition of hydride at C-10 to true reduction products, e.g.
bilirubins, as revealed by comparison of their uv-vis and tlc-data
with those of the authentic corresponding rubins (see Table I).
Reduction of verdins to rubins with borohydride in methanol has
been reported in the
-
186 W. Kufer and H. Scheer • Rubins and Rubinoid Addition
Products from Phycocyanin
R
3a R = C 2 H 3 R ' = H R = R'= 5a R = C 2 H 3 3b R = C 2 H 5 R
'=H 4a C 2 H 3 H 5b R = C 2 H 5 3c R = C 2 H 3 R ' = C H 3 4b C 2 H
5 H
4 c C 2 H 3 C H 3
C O O C H , C O O C H j
11
-
W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products
from Phycocyanin 187
literature [4, 5]. The appearance of III- and XIII- a isomers 5
and 6 besides bilirubin (4 a) and mesobili-rubin (4 c) in
methanol/water can be explained by the isomerisation ("scrambling")
reaction, which has been observed with bilirubin in moderately
basic aqueous Solutions [11]. Structure 2 a was thus assigned to
the reaction product of phycocyanin with borohydride, and since it
is a true hydrogena-tion product is should be named
"phycorubin".
Conflicting results have been reported for the reaction of
verdins with dithionite. Bilirubin has been isolated in low yield
from the reaction of sodium dithionite with biliverdin, in aqueous
al-caline Solution [16], and the reduction of a bil i-verdin-iodine
complex to bilirubin has been re-ported [17]. On the other hand, a
product different from bilirubin has been identified as well [18].
The model studies reported here support this differential
reactivity of bilindiones towards dithionite. In the neutral
pH-range studied, rubins have only been identified in low yield,
and only in Solutions con-taining methanol. In particular, rubins
have in no case been observed under the conditions used for the
reactions of PC with sodium dithionite. A product with similar
Chromatographie mobilities as the sulfite product (see Table I)
arose instead. Deri-vatives of nicotinamide are reduced by
dithionite in a complex mechanism. Addition of sulfinate, the
homolytic fission product of dithionite, has been suggested as the
first step, followed by a slower and pH-dependent elimination and a
final true reduc-tion step with HSOj rather than S 207 as the
reagent proper for hydride transfer [19]. Whereas this se-quence
eventually leads to reduction of nicotin-amide in good yields, this
is apparently not the case with bilindiones, possibly due to the
increased stability of the first addition products *. Even
pro-longed treatment of the product at p H 2.7 during workup did
not result in bilirubin formation. Struc-ture 2 b is thus suggested
for the dithionite reaction products of PC and A P C , and
analogous structures
* Caughey and Schellenberg [20] have identified sul-finate
addition products of nicotinamides by JHmr spec-troscopy. Attempts
to identify the corresponding product of biliverdin failed. 3 c in
C 2HC1 3 (0.02 M) was treated with an aqueous Solution of sodium
dithionite, and the yellow Solution formed was thoroughly washed
with water and dried. The Solution exhibited only broad Signals.
When the reaction was carried out with sodium borohydride instead,
the spectrum of the product was identical to that of bilirubin
dimethylester (4 c) (see experimental part).
for the reaction products with other biliproteins, e.g.
phycoerythrin, and phytochrome P r . They had earlier been
tentatively identified as true reduction products [2, 3], which has
to be corrected in view of the comparison with the rubins proper
obtained with borohydride. Thus, the stability of phycorubin (2 a)
upon renaturation (removal of urea e. g . by gel filtration), was
strikingly different as compared to the products obtained with
sulfite (2 c), 2-mercapto-ethanol (2d) and dithionite (2 b) [3] see
Scheme 1. The yellow colour of the borohydride product remained
unchanged, while the products with sul-fite, 2-mercaptoethanol and
dithionite were con-verted back to native PC.
The reactions summarized in Scheme 1B, can read-ily be explained
by a reversible addition to the central methine bridge, with the
equilibrium strong-ly dependent on the State of the protein. In the
denatured State with the chromophores still bound but uncoupled
from the protein, the addition-elimi-nation equilibrium is shifted
to the addition pro-duct, whereas the eduet is favored in the
native or renatured pigments even in the presence of a large excess
of dithionite. Also, the equilibrium is dif-ferentially affected by
the different environments of the chromophores of identical
molecular structure (e.g. three in PC). Reversible addition
reactions are thus a useful tool to probe these different
environ-ments [3].
The different stabilities of the native and dena-tured pigments
may originate from a different con-formational freedom of the
chromophores in the two states. Biliverdin dimethylester (3 b) and
related bilindions are rather flexible molecules, which exist in
Solution in several rapidly interconverting forms [21] of
predominantly cyclic conformation [22, 23]. The nearly identical
spectroscopic properties of denatured biliproteins make a similar
mobility like-ly for their chromophores. The comparably narrow
absorption bands of native biliproteins, as well as their high
fluorescence yield indicate that the chro-mophores are rigidly
fixed in (an extended [24, 25]) conformation. The nucleophilic
addition results in the conversion from an sp2 to sp3 hybridised
C-10, and thus necessarily a conformational change of the
chromophores. A conformational fixation could then shift the
equilibrium between the biliprotein chromophore proper, and its
addition product. We have attempted to test this hypothesis by
studying the reaction of phorcabilin-dimethylester (9) and
-
188 W. Kufer and H. Scheer Rubins and Rubinoid Addition Products
from Phycocyanin
isophorcabilin dimethylester (10), bilins held in an extended
conformation by additional bridges be-tween the pyrrole rings.
Unfortunately, however, the central seven membered ring is still
flexible enough to allow both sp2- and sp3-hybridization of C-10
without steric restrictions. Thus, 9 and 10 react with both
dithionite and borohydride as well as does biliverdin-IX
y-dimethylester 11 (see Table II).
The slow formation of denatured PC from the dithionite product
[3] after removal of the reducing agent (gel filtration, see Scheme
la) is then ex-plained by a shift in the equilibrium to the side of
phycocyanin.
According to the structure of the phycorubins obtained with N a
B H 4 as true reduction products, their properties are quite
different. The major dif-ference between the addition of
nucleophiles to and the hydrogenation of biliverdin is, that the
former reaction is in thermodynamic equilibrium, whereas the latter
h essentially irreversible under the reac-tion conditions.
Biliverdin can be reduced to bi l i -rubin only with rather strong
reductants (e.g. NaBH 4 ) , whereas the oxidation of bilirubin to
biliverdin requires high-potential quinones* [4, 14]. Recent
electrochemical studies have shown, too, that the
biliverdin-bilirubin redox couple is not an equilibrium System
[26]. The striking difference between phycorubin and the addition
products of phycocyanin summarized in Scheme 1 can readily be
explained on this basis. The yellow phycorubin chromophore remains
stable upon renaturation of the protein and removal of the reducing
agent, sodium borohydride. Moreover, the absorption maximum
remained roughly in the same position and the extinction
coefficient was the same within experimental error. The geometry of
the phyco-cyanin chromophores is profoundly changed when the
protein is renatured and v i c e versa, which is reflected by
pronounced spectral changes [25]. The absence of similar effects in
phycorubin could then indicate, that the altered chromophores do no
longer fit the binding sites, and remain — although co-valently
attached — only loosely coupled to the protein. It should be noted,
though, that the uv-vis-
* We have achieved the reoxidation of phycorubin with
benzoquinone to products identical with PC if judged from
absorption, fluorescence and electrophoretical mobilities. Details
of this procedure and the reaction of PC with quinones [27] are to
be published separately.
spectra of bilirubins [28] appear less conformation dependent
than those of biliverdins [29].
The different binding Situation of the chromo-phores in native
phycocyanin and phycorubin can also be the reason for their
different electrophoretic mobilities (Fig. 2). Phycorubin migrates
faster to-wards the anode, which could be due to an increase of the
net negative charge by the free carboxylic acid side chain(s) at or
close to the surface. The ready reaction of renatured phycorubin
with diazot-ized ethyl anthranilate leading to a product which
cannot be distinguished uv-vis spectroscopically (Amax = 485 nm)
from the denatured form, is compat-ible with this proposal. In
SDS-gel electrophoresis, PC and phycorubin migrated identically,
indicating the absence of proteolytic artefacts during the
prepa-ration of the latter pigment.
In view of the irreversible reactions of N a B H 4 , the
treatment of native PC may be used to discriminate between
thermodynamic and kinetic effects in the reactions of biliprotein
chromophores. In first order, one may assume that (i) the
chromophores react with N a B H 4 i f only their methine bridges
are acces-sible to the reagent, and that (ii) this reaction may be
slowed down by the influence of the protein, but is irreversible
once the chromophore has been re-duced. The influence of the State
of the protein on the reaction kinetics with N a B H 4 has already
been stud-ied by Crespi et a l . [45], and interpreted as a change
in accessibility of the cromophores to the reagent. A n alternative
mechanism of the protein to slow down the reaction with sodium
borohydride would be a conformational change of the chromophore.
The similar reactivity of biliverdin and isophorcabi-lin does not
prove this assumption, but indicates that a mere conformational
change without the coplanar fixation suggested for PC has no
dominant in-fluence.
The reaction of native PC with dithionite is incomplete even at
high concentrations of the re-agent. In this case the reaction is
determined by thermodynamics. Sodium borohydride reduces na-tive PC
slowly, but completely. This demonstrates, that C-10 of all
chromophores is accessible by the reagent dissolved in the aqueous
phase. Similar conclusions have been reached by Troxler [30] for
the terminal 0-1 and 0-19, and hence practically the entire
chromophore must be accessible to the aqueous phase. It requires
further work to deter-mine, whether the chromophores are actually
at the
-
W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products
from Phycocyanin 189
surface, or rather in interior hydrophilic parts of the
protein.
The possibility of the preparation of a phycobili-protein with a
rubin-type chromophore described here makes further modifications
feasible, since bi l i -rubins are accessible to a variety of
reactions. A first example is the use of the diazoreaction of
bilirubin as a novel degradation technique for biliproteins
[31].
Materials and Methods
PC was isolated from S p i r u l i n a p l a t e n s i s as
de-scribed previously [3].
P r e p a r a t i o n of P h y c o r u b i n
10.0 ml of a Solution of denatured P C (5 x 10~5 M chromophores)
in 50 mM sodium phosphate/8 M urea-buffer (containing 5 mM E D T A
and 5 mM NaN 3 ) , pH 7.5, were cooled with ice and 500 ul of a
Solution of 10 mg/ml sodium borohydride in the same buffer were
added. The Solution was left Standing for 1/2 h and then again 500
ul of the borohydride Solution were added (2.4 x 10"2 M final
concentration). With-in again 1/2 h the reaction followed
spectrophoto-metrically went to completion.
R e n a t u r a t i o n of p h y c o r u b i n
For removal of urea (and borohydride) 1.0 ml of the Solution
obtained by the reaction described above was passed through a 1.7 x
12 cm biogel P 2 column (Bio-Rad, Richmond, California) which was
previously equilibrated with 0.05 M sodium phosphate buffer, pH
7.5. The procedure was car-ried out in a cold room at 4 °C.
P r e p a r a t i o n of m o d e l b i l i n s f o r t h e P C -
c h r o m o p h o r e a n d of a u t h e n t i c r u b i n s
Bilirubin (4 a), biochemical grade, was purchased from Merck,
Darmstadt. Mesobilirubin (4 b) was prepared from 200 mg of 4 a by
catalytic hydro-genation [4, 32] (40 mg of 10% Pd on charcoal) in
20 ml 0.1 N NaOH. The reaction was followed
spec-trophotometrically, adding aliquots from the reac-tion mixture
to methanol (A m a x (4 a) = 452 nm, X M A X (4b) = 428nm). The
mixture was worked up with Chloroform after addition of
glycine/HCl-buffer, pH 2.7, saturated with ammonium sulfate. Yield:
98%. Biliverdin (3 a) was prepared from 50 mg bili-
rubin (4 a) dissolved in 50 ml dimethylsulfoxide by oxidation
with a Solution of 50 mg 2,3-dichloro-5,6-dicyanobenzoquinone in 10
ml of the same solvent in a nitrogen atmosphere [4]. After work-up
with Chloroform and water (yield: 38%), the reaction product was
purified by preparative tlc on silica using the upper phase of a
mixture of toluene/acetic acid/water 5:5:1 (v/v/v) as solvent
System. The green band (ÄF-value 0.1) was eluted with acetic acid
and worked up by partitioning between Chloro-form and water. The
Chloroform phase was washed with water and 1% aqueous NaHC0 3
-solution until neutral (yield: 26%). For crystallization the
product was dissolved in 3 ml Chloroform (containing a small amount
of methanol). 12 ml of «-hexane were added and the mixture was left
Standing at - 20 °C over night (yield: 9%).
Mesobiliverdin (3 b) was prepared by an analo-gous procedure
from mesobilirubin (4 b).
10 mg of crude (3 a) dissovled in 150 ml methanol were
esterified to the corresponding dimethylester (3 c) by treatment
with 60 ml methanol containing 20% (w/w) B F 3 [4] under reflux for
10 min in a nitrogen atmosphere. After work-up with Chloro-form and
water, the crude product was purified by tlc on silica with
chloroform/acetone 95:5 (v/v) as solvent [33]. The green zone was
eluted with acetone (yield: 85%).
Bilirubin dimethylester (4 c) was prepared from 30 mg (4 a)
suspended in 10 ml CHC1 3 by esterifica-tion with diazomethane over
night [34, 35]. The Chloroform phase was washed with an aqueous 10%
NajjCOa Solution and the product subsequently chro-matographed on
neutral A 1 2 0 3 (2x6 cm; super ac-tivity I, Woelm, Eschwege)
[36]. By-products were eluted with CHC1 3 , (4c) with C H C l 3 / M
e O H 9:1 (yield: 65%).
Octaethylbilindiones (7) and (8) were synthesized from
octaethylporphyrin [37, 38]. The products were purified by tlc on
silica with CCl 4/acetone 9:1 (v/v) as solvent.
Biliverdin-IX y-dimethylester (11): a mixture of the isomeric
biliverdin-IX dimethylesters was ob-tained by coupled oxidation
(02/hydrazine) of hemin with subsequent esterification [33].
Biliverdin-IX dimethylester was freed from the ß and 5-isomers by
tlc on silica with chloroform/acetone 97:3 (v/v) [39], and
subsequently from the a-isomer by tlc on silica with
toluene/ethylmethylketone/acetic acid 10:5:0.5 (v/v/v) [40].
-
190 W. Kufer and H. Scheer Rubins and Rubinoid Addition Products
from Phycocyanin
Phorcabilindimethylester (9) was prepared by heating
biliverdin-IX y-dimethylester (11) for 1 h at 100 °C in
dimethylsulfoxide under nitrogen [39]. Isophorcabilindimethylester
(10) was obtained from phorcabilindimethylester (9) in 20% M e O H
/ H 2 S 0 4 , the mixture being kept under reflux for 1/2 h [39].
The products were purified by tlc on silica with chloroform/acetone
8:2 (v/v) as solvent System and crystallized from CHCl 3 /«-pentane
1:25 at - 20 °C.
Mixture of the III-, IX- and XHI-a-isomers of bi l i -rubin (4
a, 5 a, 6 a) and mesobilirubin (4 b, 5 b, 6 b): bilirubin (4 a) or
mesobilirubin (4 b) were dissolved in 0.5 ml 0.1 N N a O H and
added to 5.5 ml 50 mM sodium phosphate buffer, pH 7.5, containing 8
M urea. The samples were purged with nitrogen and kept at 34-38 °C
for 2 h. The rubins were extracted with Chloroform after addition
of glycine/HCl buf-fer, pH 2.7, saturated with ( N H 4 ) 2 S 0 4 .
For prepara-tion of pure isomers of bilirubin, the mixture was
chromatographed on silica with C H C l 3 / H O A c 97:3 (v/v) [7 c]
and the products eluted with Chloroform.
The model Compounds were reacted with sodium borohydride
(Merck-Schuchardt, Hohenbrunn, p.s.) and sodium dithionite
(technical grade, Merck, Darmstadt). The reaction mixtures were
worked up by extraction with 1.5 vol of Chloroform after addi-tion
of 1.5 vol. glycine/HCl-buffer, p H 2.7, satu-rated with ammonium
sulfate in the case of free acids [5]. The Chloroform phase was
briefly dried over NaCl and after filtration on cotton, uv-vis
spectra were taken. The samples were dried in a stream of nitrogen
for tlc.
P r e p a r a t i v e s c a l e r e a c t i o n of b i l i v e r
d i n d i m e t h y l e s t e r (3 c) w i t h d i t h i o n i t
e
A Solution of 10 mg 3 c in 10 ml methanol was added to a
Solution of 50 mg sodium dithionite in 10 ml doubly glass distilled
water under a nitrogen atmosphere. After 10 min, 20 ml
glycine/HCl-buf-fer, pH 2.7 [5] were added and nitrogen was bubbled
through the Solution for further 30 min. The mix-ture was extracted
twice with 10 ml Chloroform, with some yellow pigment remaining in
the aqueous phase. Preparative tlc of the organic extracts on
silica gel (0.75 mm) with benzene/ethanol = 25:2 (v/v) yielded four
zones: 1, yellow, Rf = 0 ; II, yellow, i ? F = 0.3-0.5; III, yellow
with greenish and brownish components, R F = 0.65; IV, blue, R F =
0.70 (identified as the starting material by analytical tlc
with System "a" of Table I). Bands II—IV were elut-ed with
acetone, elution of zone I was impossible. Zones II and III were
each fractionated by column chromatography (2 x 5 cm valumina,
neutral, activi-ty super 1; Woelm, Eschwege): zone II: the main
fraction was eluted with Chloroform, a minor frac-tion with
chloroform/methanol 9:1, zone III: two fractions were separated
with Chloroform, one with chloroform/methanol 9:1, a further zone
could not be eluted with methanol.
P r e p a r a t i v e s c a l e r e a c t i o n of b i l i v e r
d i n (3 a) w i t h d i t h i o n i t e
7.8 mg biliverdin were dissovled in 50 ml hot methanol and after
cooling, added to a Solution of 500 mg sodium dithionite in 50 ml
water under a continous stream of nitrogen. 400 ml
glycine/HCl-buffer, saturated with ammonium sulfate, p H 2.7 [5],
were immediately added and the aqueous phase was extracted four
times with a total volume of 300 ml of Chloroform. The Chloroform
phase was concentrated in vacuo to 30 ml and fractionated by tlc on
silica gel plates (0.25 mm) with benzene/ ethanol = 25:2 (v/v). A
zone with green, brown and violet coloured substances with a yellow
margin re-mained at the Start. The only major zone (orange R F =
0.4) was eluted with 130 ml Chloroform to yield 0.8 mg crude
bilirubin (4 a); it was esterified with C H 2 N 2 in CHC1 3 .
Chromatography on A1 2 0 3 yielded the following fractions: I,
yellow substance eluted with Chloroform; II, main fraction, yellow
substance eluted with C H C l 3 / M e O H 9:1= yield 0.3 mg; A m a
x (CHC13) = 403 nm, Shoulder at 435 nm; identical in
cochromatography with bilirubin-dimethylester (4 c) with System a
of Table I; III, yellow-greennish sub-stance, eluted with CHC1 3
acetic acid =1:1.
*HMR-spectra of biliverdin dimethylester (3 c), its reaction
products with borohydride and dithionite, and of authentic
bilirubin dimethylester (4 c):
Preparation of the reactions products: 3 c /NaBH 4 and
3c/dithionite: A Solution of 6 mg biliverdin dimethylester (3 c) in
2 ml CHC1 3 was shaken with 1.0 ml of an aqueous Solution of 50
mg/ml di-thionite until the colour changed to yellow-green. The
CHC1 3 phase was washed with water, dried on NaCl and evaporated
under a stream of nitrogen. An analogous experiment was carried out
using sodium borohydride instead of dithionite.
-
W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products
from Phycocyanin 191
Biliverdin dimethylester (3 c): 1.80 (exo-CH 3); 2.01, 2.04,
2.10 ( e n d o - C R ^ 2.56 t, 2.93 t (8.12-C H 2 - C H 2 ) ; 3.61
(OCH 3 ) ; 5.28-6.61 (vinyl-protons); 5.94,6.00 (5.15-CH); 6.73
(10-CH).
Bilirubin dimethylester (4 c): 1.68 (exö-CH 3); 1.91, 202 ( e n
d o - C H ; 2.39 t, 2.84 t (8.12-CH 2-CH 2); 3.63 (OCH 3 ); 4.10
(10-CH 2); 4.65-6.66 (vinyl protons); 5.84, 613 (5.15-CH);
10.04,10.12,10.42,11.14 (NH).
3 c / N a B H 4 : 1.68 (exo-CH 3); 1.91, 201 ( e n d o - C H 3 )
; 2.38 t, 2.83 t (8.12-CH 2-CH 2); 3.63 (OCH 3 ) ; 4.09 (10-CH2);
4.65-6.66 (vinyl protons); 5.84, 6.12 (5.15-CH); 10.03,10.12,
10.41,11.11 (NH).
3c/dithionite: Broad Signals in the ranges 1.5-3.6 ppm and
5.1-6.4ppm; no signal detectable in the ränge 3.7-5.0 ppm.
G e n e r a l methods:
UV-vis spectra were taken with a model D M R 22
spectrophotometer (Zeiss, Oberkochen), the spectra of Compounds
9-12 with a model Superscan spec-trophotometer (Varian, Palo Alto).
x Hmr spectra were recorded in C 2 HC1 3 with a model H F X 9 0
spectrometer (Bruker, Karlsruhe). Chemical shifts in ö (ppm) are
given relative to S i (CH 3 ) 4 as internal Standard. Signals
appeared as singlets, unless other-wise noted. t = triplet. The
vinyl protons appeared as A B X System.
Tris-glycine electrophoresis was done by the meth-od of Davies
[41] as modified by Wagenmann [42], SDS-electrophoresis with the
System of Weber and Osborn [6]. Densitograms were obtained with a
type T L D 100 densitometer (Vitatron). Analytical tlc was carried
out on silica H P T L C plates and polyamide 11 F 254 plates (both
Merck, Darmstadt). Prepara-tive separations were performed on
self-made 20 x 20 cm plates covered with 0.75 mm silica H (Merck,
Darmstadt).
A c k n o w l e d g e m e n t s
This work was supported by the Deutsche For-schungsgemeinschaft,
Bonn-Bad Godesberg. We are indebted to Prof. W. Rüdiger for
continuing Sup-port. We thank Prof. R. Gautron (Grenoble) for
sti-mulating discussions and Dr. C. Petrier (Grenoble) for
aquainting one of us (W.K.) with the prepara-tion of
isophorcabilin. The visit to Grenoble was made possible by a travel
grant from the European Photochemistry Association. We thank Mrs. G
. Schild for the measurement of the *HMR spectra, Mrs. H .
Wieschoff and Mrs. C. Bubenzer for valu-able technical assistance
in the preparation of the phorcabilins and C-PC, respectively.
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