:lVERSITY OF Hgg p LIB

:lVERSITY OF Hgg p LIB

PROPERTIES AND. SUBCZLLULAR DISTRIBUTIONOF TVQ ORN I'THINE TRANS CARBAMOYLAS E S

IN THE ARGININE.METABOLISM OF SUGARCANECELL SUSPENSIONS

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISIONOF THE UNIVERSITY OF HAWAII IN PARTIAL

FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE QF

DOCTOR OF PHILOSOPHY

IN BOTANICAL SCIENCES

AUGUST 1978

By

Edward Perry Glenn III

Dissertation Committee:

Noel P. Kefford, ChairmanAndrew Maretzki

Suresh S. Patil

Edison W. Putman

Chung-Shih Tang

Ne certify that ze have read this dissertation and that

in our opinion it is satisfactory in scope and quality as a

dissertation for the degree of doctor of philosophy in

botanical sciences.

DISSERTATION COMMITTEE

ABSTRACT

Two forms of ornithine transcarbamoylase EC 2, 1. 3. 3!

were partially purified from sugarcane cell cultures. The

two forms differed in subcellular distribution, molecular

weight, and kinetic properties with respect to ornithine and

citrulline. Based on kinetic properties the mitochondrial

enzyme was assigned an anabolic role and the cytoplasmic en-

zyme a catabolic role. Only the mitochondrial ornithine

transcarbamylase was regulated by exogenous arginine in

vivo. Growing sugarcane cells were found to decarbamoylate

citrulline at approximately two times the rate that cyto-

plasmic ornithine transcarbamylase catalyzed the same re-

action in vitro. However, no evidence for the existence of

the complete arginine desiminase pathway, of which a cata-

bolic ornithine transcarbamylase is normally a part, was

found in sugarcane cells. The ce11s did not contain argi-

nine desiminase nor was citrulline detected as a product of

arginine catabolism in isotope feeding experiments. The

results with sugarcane are in contrast to those obtained

with other eucaryotes mammals and fungi! which contain a

single, mitochondrial, orniehine transcarbamoylase.

iV

CONTENTS

LIST OF TABLES

LIST OF IGURES ' e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ t t ~ t ~ eF

V i 1. iLI,ST OF ABBREVIATIONS

CHAPTER I. TEE ORNITHINE CYCLE IN HIGHER PLANTS

CHAPTER II. TWO QRNITH,INE TRANSCARBAMOYLASES INSUGARCANE CELL CULTURES

CHAPTER I,II. DECARBAMOYLATION OF CITRULLINE BYSUGARCANE CELLS

CHAPTER EV-. ENZYMES OF ARGININE CATABOLISM INSUGARCANE CELLS

CHAPTER V. CARBON DIOXIDE PRODUCTION FROM ARQINBY SUGARCANE CELLS 54~ ~ ~ ~

CHAPTER VI CONCLUSIONS e ~ ~ ~ ~ e ~ ~ t q ~ ~ ~ ~ ~ e ~ t t . ~ ~ ~ ~

TABLES t ~ ~ ~ a ~ ~ ~ ~ ~ ~ ~ ~ ~ t ~ ~ ~ ~ ~ t ~ ~ ~ ~ ~ ~ K3

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ i ~ ~ ' ~ ' ~ ~ ~ ~ 0 'P 0 ~ . 76FIGURES

LITERATURE CITED ,....,,....,,........,.....,.....,... 102

ABSTRACT .. ~ ..........,...,......,...,................ iii

LIST OP TQBLESt

Table

DISTRIBUTION QP QRNITHINE TRANSCARBANQYLASEIN SUBCELLULAR FRACTIONS ,...... ~ ~ ,...,...,, ... 64

D I STRT BUT ION QF ORN ITKINE TRAN S C AREANOYLASEAND MARKER ENZYMES IN SUBCELLULAR FRACTIONS -,.-. 65

EFFECT OF pH ON OTC'S AT CONSTANT ORNITHINECONCENTRATION IN DIRECTION OP CITRULLINESYNTHES I S ~ ~ ~ ~ ~ ~ t ~ ~ ~ ~ 66

EFFECT OF pH ON OTC ACTIVITIES AT NON-SATURATING ORNITHINE CONCENTRATIONS ...-........ 67

IV

EPFECT OP EXOGENOUS ARGININE ON CYTOPLA'SMICAND MITOCHONDRZAL OTC'S IN SUGARCANE CELLS

DISTRIBUTION OF RADIOACTIVITY IN CELLS FED1 C-CITRULLINE FOR FIVE HOURS

VI69

OTCC AND OTCM ACTIVITIES IN CITRULLINE ANDORNITHINE SYNTHESIS

VII70

ABSENCE OF ARGININE DESIMINASE AND ARGINASEFROM SUGARCANE CELL-FREE EXTRACT .... ~ ... ~ ~

VIII71

ARGININE DECARBOXYLASE AND UREASE IN SUGARCANECELL, PROTEIN, CONCENTRATES .................... 72

DISTRIBUTION OF RADIOACTIVITY IN STOCK GUANIDINO-14C-ARGININE AND IN CELLS INCUBATED FOR ONE HOURWITH GUANIDINO � C-ARGININE . ~ ~ ..... ~ ........... 74

XI

PRODUCTION OF CARBON DIOXIDE BY SUGARCANE CELLSFROM UREA, THE GUANIDINO CARBON OF ARGININEv ANDPROM ALL SIX CARBONS OP A'RGININE ............... 75

XII

DISTRIBUTION OF RADIOACTIVITY IN SUGARCANE CELLSINCUBATED IN GUANIDINO- C-ARGININE .... ....... 73

LIST QF FIGURES

~Fi ere

THE ARGININE DESIMINASE PATHWAY ..-.-... ....--. 82

THE AGMATINE PATHWAY ......,.....,......,. . ., 83

ORNITHINE BIOSYNTHESIS IN CHLAMYDOMO'NAS ~ o ~ i ~ ', e ' 84

ELUTION OF QTC AND QTC ON DEAE-CELLULQSE ,... 85

ELUTION QF A MIXTURE OF OTC AND OTC ON

DEAE-CELLULOSE ...,...,.....,,,...,.....,...,.,� 86

ELUTION OF A MIXTURE OF OTC AND OTC ONC

AGARO SE GE'L ~ ~ ~ ~ ~ e ~ ~ ~ ~ ~ ~ e ~ ~ e ~ e ~, q ~ 87

MOLECULAR WEIGHT ESTIMATIONS FQR OTC AND QTC . 88

EFFECT OF pH ON THE FORW'ARD OTC REACTION FOROTC AND OTC ...,.......,...,...,...;,....,... 89

1O

EFFECT OF pH QN THE REVERSE OTC REACTIQN FORQTCC AND OTC ''''''''' '''''''' ' '' '' ' 9Q

EFFECT QF ORNITHINE ON OTC AND OTC IN CITRUL-

LINE SYNTHESIS AT pH 8 0 AID 8.5 .-.....-.....-, 91

13a DOUBLE RECIPROCAL PLOT OF SATURATION OF OTC

BY ORNITHINE .......,.....,. ~ .,,...,....., ..., 92

13b DOUBLE RECIPROCAL PLOT OF SATURATION QF OTCMBY ORNITHINE .......,.........,,....,...,..., , 93

14a DOUBLE RECIPROCAL PLQT OF SATURATION OF OTC

BY CARBAMOYLPHOSPHATE .....,............,....., 94

DOUBLE RECIPROCAL PLOT OF SATURATION OF OTC

BY CARBAMOYLPHQSPHATE ... ~ .... ~ . ~ ~ 95

15a DOUBLE RECIPROCAL PLOT OF SATURATION QF OTC

BY CITRULLINE ........,........ ~ ...,.........,

15$ DOUBLE RECIPROCAL PLOT OF SATURATION QF OTCMBY CITRULLINE ........,...,,,,......,....,...,,, 97

16 DECARBAMOYLATION QF CITRULLINE BY SUGARCANE

CELLS IN A CONTINUOUS FEEDING EXPERIMENT ,...,. 98

~pp, e

THE UREA CYCLE IN MAMMALS ...,,,,....,...,...,, 80

MODEL OF THE ORNITHINE CYCLE IN PLANTS -...,... 81

~Pa e

DECARBAMOYLATIQN OF CJTRULLINE IN A PULSE-

LABEL EXPERIMENT ...... ~ .....,,............,,, . 99

INSTANTANEOUS 'RATE OP CARBON DIOXIDE PRO-

DUCTION AT HAIF. HOUR INTERVALS IN PULSE-LABEL

EXPERIMENT PITH CITRULLINE ,.....,....,.... ~ ..., 1QQ

EVOLUTION OF CO2 FROM GUANIDINO- C-ARQININE14 14

BY SUGARCANE CELLS ....,....,.....,.. .,......,, 10,

LIST OF ABBREVIATIONS

ornithine transcarbamoylase.

succinic dehydrogenase

OTC

SDH

glucose-6-phosphate isomeraseGP I

POPOP

2, 5-diphenyloxa z o le

I, 4-his L2 '5-phenyloxaz olyl! j ! benzene

CHAPTER I

The Ornithine Cycle in Higher Plants

The ornithine urea! cycle is a metabolic pathway that

manufactures the amino acid arginine and urea �1! Figure

1!. In the cycle, ornithine is converted to arginine with

citrulline and argininosuccinate as intermediates; arginine

is then hydrolized to regenerate ornithine and to produce

the end product urea �9!. The complete ornithine cycle is

usually associated with animals that excrete urea as a ni-

trogen waste products For example, in the liver of mammals

the cycle generates 95% urea and only 5% arginine �9!-

Since higher plants do not excrete nitrogen, they might be

expected to contain only the portion of the cycle that pro-

duces arginine. In the last twenty-five years, however,

there have been numerous reports that many plants have a

complete ornithine cycle that is similar in operation to

the mammalian ornithine cycle e.g. 5,13,88!.

The plant ornithine cycle is thought to differ chiefly

by the inclusion of urease, which degrades urea to carbon

dioxide and ammonia as rapidly as it is produced. The func-

tion of the cycle in plants is apparently to recycle nitro-

gen stored in the arginine molecule. Thus, ammonia

liberated from urea is not excreted but is reassimilated by

reactions that require ammonia as a substrate, such as glu-

tamine synthesis Figure 2!. Evidence in support of this

theory will be reviewed here. Three lines of evidence are

cited in support of a complete ornithine cycle in plants

88!: the existence of the intermediates of the cycle in

plant tissues, the interconvertability qf radioactive orni-

thine, citrulline, and arginine, and the occurence of the

enzymes of the cycle.

Existence of the Intermediates

The detection of ornithine, citrulline, argininosuccin-

ate, and arginine provided the first evidence for the oper-

ation' of an ornithine cycle in p1ants. Although endogenous

concentrations of these intermediates may be 'below detect-

able levels, a broad survey shows that each compound ac-

cumulates in the cell sap or vascular fluid of one or more

plants. Ornithine rarely accumulates normally!, but it is

a major constituent of the soluble amino acid pool of bean

plants infected with Pseudomonas pathogens 95!, which pro-

duce an exotoxin that inhibits host ornithine transcarba-

moylase 83,136!. Citrulline occurs normally in the

pressed sap of watermelon �45!, and it is the major nitro-

gen compound in the xylem fluid of some trees 88,143!.

Argininosuccinate is less common but has been detected in

germinating seeds of white lupin �0!, pea �2!, and Jack

bean �46!. Arginine is ubiquitous; it is a protein con-

stituent and also occurs in the free state in the storage

tissues of many plants, including species of marine algae

�1,2! mosses �2!, gymnosperms �,72!, and angiosperms

88! .

Operation of the complete ornithine cycle generates

urea. However, early reports of high concentrations of

urea in plants may have been due to an artifact of the iso-

lation procedure which produced urea, from allontoi c . acid

during extraction �37!. By modern methods of extraction

urea is either found to be absent from plant tissues or

present only at very low concentrations �3!. This has

been rationalized by citing the high urease content of most

plant tissues 9!.

Carbamoylphosphate is another putative intermediate in

the cycle that is rarely observed. Its half-life in aque-

ous solution is only 38 minutes �8! which precludes its

by rapid extraction and by histochemical techniques �1!.

Interconvertability of the Intermediates.

As carbon-14 labelled substrates became available, the

operation of the ornithine cycle was studied in more de-

tail. The synthetic portion of the pathway has been con-

firmed in many plant tissues by demonstrations that

14 14administered C-ornithine and C-citrulline give rise to14

C-arginine, among other products �2,13,20,25,36,37,40,

43,56,75,88,138!. The participation of carbamoylphosphate

was indirectly indicated by the fact that CO fed to14

wheat leaves in the dark was incorporated into the guanidino

carbon of arginine �!. The catabolic portion of the path-

way has been studied by infiltrating plant tissues with

14u»f ormly-labelled or guanidino-labelled C-arginine.

Generally arginine was catabolized to the glutamic acid

family of products, with ornithine aa an intermediate �,6,

13,25,36,37,40,43,53,75,77,119,120,121,122,123,128,147!.

14 14 14Sometimes C-arginine produced C-citrulline and C-

argininosuccinate �2,36! which were thought, at first, to

arise from a reversal of the ornithine cycle �2!, but are

now thought to result from ornithine reentering the cycle

.14 15�3!. From studies with C-urea �48! and N-urea �4!,

it has been concluded that urea is degraded by urease prior

to reassimilation of its carbon and nitrogen atoms.

14ln the degradation of C-arginine by plants, labelled

urea is seldom observed. Small amounts were formed in white

14lupin seedlings infiltrated with guanidino- C-arginine

�3!, but in a similar experiment with watermelon seedlings,

14employing uniformly-labelled- C-arginine, the resulting

labelled urea had only one-fifth the specific activity of

the resulting labelled ornithine �3!. The usual absence of

urea may be interpreted to indicate the existence of the

arginine desiminase pathway Figure 3! which also produces

ornithine from arginine, but with citrulline and carbamoyl-

phosphate rather than urea as intermediates. The pathway

is unique because it produces ATP from carbamoylphosphate

catabolism. It is found in many bacteria �,66,82,84,124,

150! and in Chlorella �03! and Chlamydomonas �30!, but

its existence in higher plants has apparently not been

investigated.

pathway whi'ch produces putrescine Figure 4!

In some plants, arginine also gives rise to

the agmatin.e

�5>114,115!

a variety of guanidines �,88! and in sugarcane cells free

guanidine is produced �5!. The ornithine cycle and these

alternate pathways can co-exist in the same tissue �4,65!.

Enz mes of the Ornithine Cycle in Plants .

The most recent phase of research on the ornithine

cycle involves isolating the individual enzymes and in de-

termining their kinetic and regulatory properties and their

subcellular distribution.

While not considered part of the mammalian ornithine

cycle, the enzymes producing ornithine from alpha-keto-

glutarate may be part of the plant ornithine cycle. In

bacteria �4,150!, fungi �44!, and algae �8,70,125,131,

135!, ornithine synthesis proceed.s via acetylated inter-

mediates Figure 5! which are apparently not involved in

mammals �10!. The function of the acetyl group may be to

earmark a pool of glutamate for arginine biosynthesis or to

protect the alpha-amino group as an organic chemist would

do when synthesizing ornithine in a 1aboratory �1!- The

existence of an acetylated pathway in higher plants rests

partly on indirect evidence because not all of the enzymes

have been demonstrated. N-acetylornithine has been

14 14C-arginine that is not catabolized to C-ornithine

may be incorporated into protein �4,36,:64! or it may enter

detected in plant t issue �3!, and a cell, f ree extract

from cultured, rose cells converted N-acetylornithine to

ornithine �4!. Tn a more recent study, the enzymatic for-

mation of N-acetylglutami.c acid was demonstrated in a num-

ber of flowering plants �1!. In all the plants tested,

arginine was an inhibitor of a,cetyl-CoA-glutamate trans-

acetylase, which is the first enzyme in the pathway and

thus a logical control point for arginine biosynthesis. A

mitochondrial location for ornithine synthesis is supported

14 14by the observed conversion of C-acetate into C-orni-

thine in isolated, mung-bean mi,tochondria �0!.

The enzymes producing arginine from ornithine, carbon

dioxide, and ammonia have all been purified, at least par-

tially, from higher plan.ts. Carbamoylphosphate synthetase

from legumes �6,78,79,80! resembles the bacterial enzyme

in that it is regulated by allosteric interactions with

both ornithine and pyrimidine nucleotides 80!, but there

is no evidence for two pathway-specific carbamoylphosphate

synthetases for arginine and pyrimidine synthesis as found

fungi �1! and mammals �9!. As with fungal carbamoyl-

phosphate synthetase and pyrimidine-specific carbamoyl-

phosphate synthetase from mammals, glutamine appears to be

the natural donor of the amino group �6! although, in pea,

ammonia can serve with 30% efficiency �9!.

Ornithine transcarbamoylase has been purified from pea

�7! and studied in crude extracts from several other

species 8,18,26,49,118,136!, There is evidence for multi,�

ple forms of ornithine transcarbamoylase occurring in apple

�18! and pea �6!. Isolated bean mitochondria incorpo-

14 14 . . . 14rated CO or C-ornithine into C-citrulline, indi-2

cating a mitochondrial location of both carbamoylphosphate

synthetase and orni,thine transcarbamoylase �0!� but ex'-

tracted ornithine transcarbamoylase from bean was 80 to 9QX

cytoplasmic �9!. Thi.s evidence suggests that there may be

separate ornithine transcarbamoylases in the cytoplasm and

mitochondria of plant cells.

Argininosuccinate synthetase and argininosuccinate

lyase have been separated and partially purified from

germinating pea seedlings �04,108!. Argininosuccinate

lyase has also been studied in bean 94! seedlings. Like

carbamoylphosphate synthetase, argininosuccinate synthetase

is an allosteric enzyme; it is inhibited by arginine and

responds to an adenylate energy charge in the incubation

mixture in vitro �05!. A specific protease has been re-

ported to regulate the level of the synthetase in soybean

cell cultures �06,107!. The intracellular location of the

synthetase and lyase have not been determined in plants,

but in fungi �49! and mammals �1,32,35! they are cyto-

plasmic.

The catabolic enzyme arginase completes the ornithine

cycle in mammals and it has now been reported from several

plants �6,48,74,120,140!. Arginase in plants appears to

be mitochondrial �8!; in fungi �49! and mammals �2! it

is cytoplasmic. A mitochondrial arginase occurs also in

the kidney cells of birds but it is thought to have only

limited activity because birds do not excrete urea �9!.

In addition to arginase, most plants contain urease.

In fact, they provide the. most abundant source of the en-

zyme, which was first crystallized from Jack bean seeds to

provide evidence that enzymes are proteins �32,133!.

Urease activity is especially high in legume seeds, where

it may account for 0.15% of total seed protein �33!, but

it is also present throughout the mature plant 89,133,

151!. The effectiveness of urea applied as a foliar ferti-

lizer indicates activity in leaves �4!. In bean cotyle-

dons, urease is contained in extracellular vesicles �3!.

The exact function of urease in plants is still debated

89!. While it is now most often considered a member of

the ornithine cycle, it has also been considered a trans-

carbamoylase which dons to water in vi'tro but to an unknown

acceptor in 'viva �11!, In support of this theory is the

18observation that 0 f rom phosphate is incorporated into

carbon dioxide in the degradation of urea, which makes

carbamoylphosphate likely as an enzyme-bound intermediate.

Urease has also been suggested to be a salvage enzyme for

soil urea deposited by ureotelic organisms 89! . Urease

may also act. on urea produced internally from chlorophyll

and pyrimidine cat.abolism 9!. A final suggestion is that

the enzyme has no as yet discoverable function �15!.

Ornithine catabolism begins with its conversian to glu-

tamic semi-aldehyde, catalyzed by arni,thine txansaminase

L-ornithine: 2-axaacid aminotransferase!. The enzyme has

been purified ta homagenei.ty fram squash �8! but there is

evidence for separate cytoplasmic and mitachandrial forms

in peanut �7! and Cu'curbita maxima �23! cotyledons. The

enzyme has also been found in mung bean ll! and sunflower

�13! mitochondria. Enzymes that further catabolize glu-

tamic semi-aldehyde have been identified, in crude extracts,

in peanuts �7!.

Origin and Function of the Plant Ornithine Cycle.

Despite their frequent comparison e.g. 5,13,88!, the

plant and animal ornithine cycles are really not closely

related in origin or function. Except for urease, the en-

zymes of the cycle occur widely even in such non-ureotelic

organisms as bacteria, molds, algae, higher plants, sharks,

reptiles, and birds �9!. The enzymes of the cycle must

therefore predate in origin the first ureotelic organisms,

which may have been primitive fish. In modern organisms,

ureotelism is found mainly in mammals �9,29,59! which must

excrete dissolved urea rather than solid uric acid because

they lack a cloaca for excretion egg-laying mammals such

the platypus are uricotelic! �9!. The enzymes in

p»nts are probably related to bacterial enzymes because of

11

similarities in ornithine synthesis and in their carbamoyl-

phosphate synthetases and because the amino acid metabolism

of plants is, in general, similar to bacterial systems �9!.

The synthetic portion of the ornitkine cycle has the

function, in autotrophic organisms, of providing argin.ine

for protein synthesis. However, arginine has other meta-

bolic roles as well; it is a substrate for the production

of arginine phosphate and creatine phosphate, both involved

in muscle c.ontraction �9!, and also gives rise, via putre-

scine, to the polyamines which are indispensible cell-

division factors in all organisms studied, including plants

�14!. In the beginning of life, arginine may not have

been a protein constituent at all. Jukes suggests that it

is an "intruder in the evolutionary process" which became

inserted acciden.tally into protein, in place of ornithine,

which was a protein amino acid in early organisms �1,42!.

Then the role of ornithine as a basic amino acid was taken

over by lysine, while arginine was retained in protein

using the ornithine codons! because it filled special

functions' flukes cites as evidence the low frequency of

arginine in protein �.2X! compared to the 10.7X expected

from the number of synonymous codons for arginine. For the

other protein amino acids there is good correspondence be-

tween frequency in protein and the number of synonymous

codons for each.

The catabolic portion of the cycle serves varied func-

t~ons in non-ureotelic organisms, In fungi, arginine

12

exists to salvage exogenous arginine; in sharks it produces

urea to maintain the blood isotonic with seawater; in birds

it may produce ornithine to detoxify benzoic acid to orni-

thuric acid. �9!. The most likely hypothesis for the oc-

currence of arginase and urease! in higher plants is that

they recycle nitrogen stored in arginine. This theory has

been advanced in several forms e.g. 17,122! which can be

combined into a simple model based on nitrogen availability

Figure 2!.

Model of the Ornithine C cle in Plants.

When soil nitrate or ammonia are abundant nitrogen is

incorporat.ed inta arginine and other nitrogen-rich amino

acids, which may be used in protein synthesis or stored

for later use. Arginine is well suited to store amino

groups because it is 32% nitrogen, all of which can be re-

moved without disrupting the carbon skeleton. In contrast

to the amides, glutamine and asparagine, which are also

considered to be nitrogen reserve compounds, arginine re-

ceives its amino groups secondarily, by transamination and

transcarbamoylation, rather than by direct utilization of

ammonia. When soil nitrogen becomes scarce, arginine is

broken down by arginase and urease or perhaps by the argi-

nine desiminase pathway! to yield ornithine, carbon diox-

ide, and ammonia. The ammonia is then reassimilated by

reactions that require ammonia as a substrate. Ornithine

13

can be further catahqlized to alpha .ketoglutarate tq yield

two more amino groups which are redistributed by trans-

amination. Alpha-ketoglutarate. can enter the citric acid

cycle where its breakdown will yield energy. The above

model is consistent with the isotope feeding experiments

which were described earlier. A realistic model must

recognize that the cycle is dependent not only on the

availability of soil nitrogen but also on the developmental

stage of the plant.

Evidence that Arginine is a Nitrogen Stora e Cpm oundin Plants.

To be considered a storage product a compound must be

observed to accumulate in resting tissue and to be con-

sumed in rapidly growing tissue �7,126!. Arginine meets

these criteria in cells grown in tissue culture �5,27,127,

139! and in the life cycle of entire plants �5,30,37,40,

l09,119!.

In carrot and potato cell suspensions the soluble ni-

trogen pool was found to be much larger in resting than in

proliferating cultures ?27!. The chief constituents were

the amides and arginine. Arginine especially, dropped in

concentration when rapid growth was induced, either by sub-

culturing or by adding needed growth factors. Ginko,

Pollen-cell suspensions and Jerusalem-artichoke, callus

cultures showed similar phenomena with respect to arginine

.,�>»139!. Arginine is also important in the life cycle of

14

intact artichoke plants, which accumulate it during winter

dormancy and consume it during rapid growth in the spring

�5!. Apple trees, too, accumulate arginine during the

winter l4,36,37,77,138!, when it is stored in the stem.

Some of the nitrogen in arginine comes from products in the

leaves translocated before abscission and some comes from

uptake from the soi1 throughout the winter �7!. Balance

sheets of the nitrogen content of the trees show that al-

though arginine rapidly disappears in early spring, over

99% of its nitrogen is retained in nitrogen-containing com-

pounds needed for cell division and growth �7!. Xxperi-

14ments with C-arginine support the role of arginase and

14urease in recycling the nitrogen because C-ornithine but

14again no C-urea! is observed as a transient breakdown

products Eventually most of the label is recovered as

14 C02, indicating respiration of the carbon skeleton in the

citric acid cycle �6,37,138!

In such legumes as white lupin �0! and Pisum �09!, as

well as the cucurbit pumpkin �19-123!, arginine, stored in

the cotyledons, accounts for up to 40/ of total seed nitro-

gen. It occurs in the free state in some seeds but is more

often found in storage proteins; in fact, arginine is

usually the most abundant amino acid in seed, storage

proteins �21!. The proteins are synthesized durin,g the

maturation of the seed on the parent plant. Arginine is

synthesized in the cotyledons from precursors imported in

15

the phloem �3! ~ ln white 'lupin, asparagine is the maj or

nitrogen donor �0! although glutamine may be more impor-

tant in other species �8!. Arginine itself is not a major

constituent of the phloem sap of most plants �8,88!.

When the seeds germinate, special hydrolases digest the

storage proteins to release free amino acids, most of which

are transported to the growing plant axis �3,40,122!. Ar-

ginine however, is largely degraded in situ. For example,

in pumpkin cotyledons infiltrated with uniformly-labelled

14 14C-arginine, over half the label is recovered as C02

�21!, The degradative pathway proceeds via ornithine, as

in apple trees. The fate of the amino groups released from

arginine has not been traced with certainty, but it is

known that in pumpkin over 99/ of the nitrogen is retained

by the developing plant �21!.

An interesting variation on the theme of arginine as a

nitrogen reserve occurs in some legumes that store the ar-

ginine-analogue canavanine �!. It accumulates in the free

state in the seeds and is metabolized by a canaline-urea

cycle that is distinct in enzymology from the plants' nor-

mal, ornithine cycle 92!. Canavanine is toxic to preda-

tors and appears to play the two roles of nitrogen storage

and protection in the plants in which it occurs 93!.

«gulation of the Plant Ornithine Cycle.

General strategies of regulation are apparent in the

arginine metabolism of some organisms. In mammals, the

16

ornithine cycle is largely regulated by ammonia �0,96! in

keeping with the role of detoxifying ammonia to urea. In

microorganisms, arginine synthesis is generally inhibited

by exogenous arginine, which may also induce arginine-

rose cells grown insynthetic pathway in higher plant.s. In

defined liquid medium exogenous arginine inhibited endoge-

nous synthesis �3,24!. The experiments involved measuring

14incorporation of label from C-glucose into arginine in

the presence and absence of an external arginine supply.

The time scale of the experiments did not allow a conclu-

»on as to whether allosteric mechanisms, or repression,

degrading enzymes �1,34,130,150!. The mechanisms are

species-specific and includ.e; induction, repression, allo-

steric control, and compartmentation of enzymes and sub-

strates within the cell �50!. In. Chlam domonas, regula-

tion is controlled both by ammonia and by arginine �34!.

Ammonia represses the catabolic enzyme arginine desiminase

�30! while arginine inhibits the' synthetic pathway by

allosteric inhibition of N-acetylglutamate phosphotransfer-

ase at the beginning of the pathway and repression of

argininosuccinate lyase at the end of the pathway �31,135!

A similar regulatory strategy might be expected in higher

plants because of the two roles of arginine in ni,trogen

storage and protein synthesis. So far, however, no over-

all strategy has been discovered.

There is some evidence that arginine controls the bio-

were responsible for the effect, nor did they indicate

which steps in the pathway were under regulation. It is

known that in a variety of higher plan.ts, arginine is an

allosteric inhibitor of acetyl-CoA-glutamate transacetylase

at the beginning of the pathway �1!, and in soy bean

at the end of thecells �06!, argininosuccinate synthetase

pathway. No enzyme from the biosynthetic pathway in a

higher plant has been found to be subject to repression by

arginine.

How are plants able to accumulate arginine to high con-

centrations without prematurely shutting down the synthetic

pathway? Storage in the vacuoles, as occurs in yeast �50!

�42,149! is one possible answer, as is theand

sequestering of arginine into storage proteins as occurs in

seeds. For Chlam domonas, it has been suggested that argi-

expected that the synthetic part of the pathway should be

present during maturation and absent during germination,

while the opposite is expected of the. catabolic enzymes

«ginase and urease. In pumpkin, arginase levels in the

cotyledons were lowest in the maturing seed and rose after

nyl-tRNA is the. true co-repressor of the synthetic path-

way, which would permit free arginine to accumulate in the

cell �31,135!.

Regulatory studies concerned specifically with the oper-

ation of the ornithine cycle in nitrogen storage have been

undertaken in legume and cucurbit seeds. In theory, it is

18

germination �20!. The findings were confirmed in Vicia

faba seedlings, which showed in addition An inverse rela-

tion for ornithine transcarbamoylase; it was highest in

activity during maturation and levels dropped several-

fold after germination �8,49!. But other enzymes of the

cycle are antithetic. Urease, which would be expected in

highest activity after germination, was at its highest

level in the dry seed in citrullus �51! and Jack bean

�01! and no ontogenic relationship between urease activity

and arginine degradation was found in either plant. The

synthetic pathway has been demonstrated to operate through-

out germination in the cotyledons of pumpkin �21!, pea

�04,108!, and bean seeds �2,13,40!, even as the cotyle-

dons wither �21!.

Does Ornithine Re-enter the Synthetic Pathwa ?

There is nothing in the model of the plant ornithine

cycle presented here Figure 2! to suggest that catabolic

ornithine actually re-enters the cycle, as it does in

mammals. In fact the opposite is expected since ornithine

is constantly removed to form the glutamic acid family of

produc.ts, Each molecule of catabolic ornithine that re-

enters the cycle in plants would consume three equivalents

ATP in the apparently needless resynthesis of arginine.

similar regulatory problem confronts bacteria and fungi

that consume arginine and they have evolved several

19

mechanisms to prevent the recycle,ng of catabolic ornithine.

Bacteria that have the arginine desiminase pathway of ar-

ginine degredation have separate anabolic and catabolic

ornithine transcarbamoylases under separate metabolic regu-

lation with respect to arginine �24,150!. The anabolic

enzyme is repressed by arginine, so it is absent when exo-

genous arginine is available. In eucaryotes, repression

may be too slow to allow an adaptation to a changing supply

of arginine because the genes are susceptible to control

only once in each cell cycle �5!, so allosteric mecha-

nisms must also operate. In yeast, ornithine transcarba-

moylase is inactivated by complexing with arginase in the

presence of high cell arginine levels �50!. In N r'

�1,149,150!, ornithine transcarbamoylase is localized in

the mitochondria while catabolic ornithine is contained in

cytoplasmic storage organelles.

There is no evidence as of now that any of the above

mechanisms operates in higher plants, but their existence

in other organisms suggests that prevention of the re-

cycling of ornithine is a general feature of arginine

metabolism in nonureotelic organisms.

Conclusions

The steps in the arginine metabolism of higher plants

are similar to those of most other organisms. Where differ-

ences exist, plants tend towards bacterial rather than

20

mammalian enzyme systems. Thus in higher plants as in

bacteria ornithine is synthesized from glutamate via ace-

tylated intermediates, and there is a single carbamoyl-

phosphate synthetase to provide substrate for arginine and

pyrimidine synthesis. plants are also similar to bacteria

in the production of putrescine by the agmatine pathway,

which in mammals is produced by ornithine decarboxylation.

Arginine catabolism in plants proceeds through arginase and

urease, and perhaps also through arginine desiminase and

catabolic ornithine transcarbamoylase, enzymes that are all

found among bacteria. On the other hand only arginase is

found in mammals'

Ornithine is an intermediate in both the synthesis and

catabolism of arginine but it is probably incorrect to con-

clude that plants contain an ornithine cycle similar to

that of ureotelic organisms. Nore likely, plants prevent

catabolically derived ornithine from entering the arginine

biosynthetic pathway by regulatory mechanisms such as are

found in other non-ureotelic organisms. Such mechanisms in

microorganisms generally center on ornithine transcarbamoy-

lase.

Nevertheless, the "ornithine cycle" of highe.r plants

plays a clear-cut role in their nitrogen economy, besides

providing arginine for protein synthesis. As the amino

«id with the highest ratio of nitrogen to carbon, arginine

utilized as a nitrogen reserve compound in many plants,

21

especially legumes and temperate-zone trees, The synthetic

portion of the cycle predominates over the catabolic por-

tion at some points. in the life cycle of .these plants, and

arginine accumulates in vacuoles and storage proteins,

Eventually, stored arginine is released into the general

metabolism where its degradation to alpha-ketoglutarate re-

leases four equivalents of reduced nitrogen to the plant.

Plants appear to be unique in the efficiency with which

the released nitrogen is retained during arginine catabo-

lism. In other organisms it is largely excreted, either in

ammonia or urea. Plants are known to contain enzymes that

efficiently fix ammonia but it is not known which enzymes

are coupled to the ornithine cycle. Glutamine synthetase

GOGAT! is considered to be important in fixing ammonia

produced in nitrate reduction but it is localized in plas-

tids �9! while the enzymes of arginine catabolism are

mitochondrial arginase! and cytoplasmic urease!. Sepa-

rate forms of glutamate dehydrogenase are located in the

cytoplasm and mitochondria of plant cells �9! and either

one or both could be coupled to the ornithine cycle.

Coordination of the anabolic and catabolic portions of

the cycle is tied to the developmental stage of the plan.t.

The regulatary mechanisms by which the coordination is

achieved, however, are still unknown. Discovering such

mechanisms will provide research goals far thase interested

the regulation af nitrogen metabolism during growth and

22

development. Progress will come from studies which probe

the regulatory and kinetic properties of individual enzymes

of the cycle, and their temporal, and spatial, organization

inside cells and tissues.

Statement of the Research Problem.

The research to be reported concerns the properties,

subcellular distribution, and biological functions, of two

forms of ornithine transcarbamoylase from sugarcane cells,

grown in liquid suspension culture. As already noted,

multiple forms of ornithine transcarbamoylase have been re-

ported from apple �18! and pea �6! tissues, and activity

has been found in both the cytoplasm �9! and mitochondria

�0! of bean cotyledons. The evidence challenges a general

theory �1,39! for the control of transcarbamoylations in

eucaryotes, which holds for a single, mitochondrial, orni-

thine transcarbamoylase. On the other hand, multiple forms

of the enzyme are common in bacteria �50!. It is now

necessary to determine the biological meaning of multiple

forms of ornithine transcarbamoylase in higher plants.

It should be kept in mind that there are two points of

view concerning the function of multiple forms of an en-

zyme. One theory holds that' all forms are of functional

significance �,91,152!, while the other holds that per-

haps 99 out of 100 are simply examples of "evolutionary

noise" �4,45,55!. The research reported here started

23

from the premise that two ornithine transcarbamoylases in

plants are functionally distinct.

A hypothesis to explain their function in sugarcane

cells can be con.structed from the mitochondrial location of

anabolic! ornithine transcarbamoylase in fungi �17! and

mammals �9!, and the existence of separate anabolic and

catabolic forms in bacteria that contain the arginine

desiminase pathway 86,150!. The hypothesis states that

g c , !

tablished that ornithine and citrulline are intermediates

arginine production, so a role for an anabolic ornithine

transcarbamoylase was assumed. Given that both forward and

separate cytoplasmic and mitochondrial enzymes catalyze

citrulline decarbamoylation and citrulline synthesis, re-

spectively. Citrulline decarbamoylation is a step in the

arginine desiminase pathway, while citrulline synthesis is

part of the arginine synthetic pathway.

Three predictions of the hypothesis were tested. First,

there should be physically distinct, kinetically special-

ized, forms of ornithine transcarbamoylase in the cytoplasm

and. mitochondria. Second, citrulline decarbamoylation

must occur in whole cells as well as cell-free extracts.

Third, arginine desiminase must be present to provide sub-

strate for a cataboI.ic ornithine transcarbamoylase. These

three criteria have been used to establish a role for a

catabolic ornithine transcarbamoylase in bacteria �.50!.

Previous studies with cultured su arcane elis 33 64 es�

24

reverse reactions occur in viva, it must still be decided

which enzymes act in which pathway. Genetic experiments

have been used to decide this question for bacteria �50!,

but in the present work with sugarcane tissue cultures only

indirect evidence was available.

Sugarcane cells were chosen for the experiments because

they previously were found to carry out almost all the re-

actions in their arginine metabolism that characterize

higher plants, including arginine synthesis �3,65! and

degradation of arginine to glutamic acid �5!. A reaction

the cells lack is decarbamoylation of 5-carbamoylputrescine;

this characteristic was useful in measuring citrulline de-

carbamoylation in whole cells, since the two reactions may

be confused. Cell cultures have been used in other studies

of amino acid metabolism in plants and their advantages and

limitations have been fully discussed �29!. Preliminary

experiments showed that sugarcane cells contained ornithine

transcarbamoylase activity in both cytoplasmic and mito-

chondrial cell fractions, and in fact provided a much higher

yield of mitochondrial enzyme than reported from bean �9!.

The following chapters report research results on the

three parts of the hypothesis that were tested. Chapter II

d.escribes the two ornithine transcarbamoylases in sugarcane

cells. Chapter III presents results on the decarbamoyla-

tion of citrulline by whole cells. Chapters IV and V exam-

ine the pathways of arginine catabolism in cell-free

25

extracts and whole cells, with the primary goal of identi-

fying the arginine desiminase pathway. The findings are

interpreted in light of the starting hypothesis in Chapter

VI.

26

CHAPTER II

Properties and Subcellular Distribution of Two

Partially Purified Ornithine Transcarbamoylases

from Sugarcane Cell Cultures

27

Introduction

Nultiple OTC's. ornithine tranacarbamqylases! in bac-

teria represent separate anabolic and catabolic forms under

separate regulatory control with respect to arginine �24! ~

Evidence is presented here that two OTC's exist in sugar-

cane cell suspensions. They differ in their subcellular

locations and in many of their physical and kinetic proper-

ties. Cell levels of the two forms also respond different-

ly to added arginine.

Materials and Methods

Cell Culturin . Unless otherwise noted, sugarcane

cells variety H50-.7209! in suspension culture were grown

in a stock medium containing yeast extract and supplemented

with 300 pCN arginine �6!. Cells from the stationary phase

of growth were harvested by suction and washed three times

with distilled water.

Preparation of mitochondria. One to 5.0 gfw cells were

ground by mortar and pestle in the cold, using a high-

ionic-strength buffer �. Manning, private communication!

consisting of 0.5M sucrose, 0.5N KH PO , 0.42N Tris, lmN

EDTA, and .075/ bovine serum albumin pH 7.2!. The initial

grinding was done in one ~olu~e w/v! of the buffer, and

the resulting homogenate was diluted 1:10 with a buffer

2containing 0. 3M sucrose, Q. 1M KH PO, and 0. 08M Tris pH

7-2! before filtering the homogenate through 4 layers of

28

cheesecloth. The filtrate was centrifuged for 5 minutes

at 250 x g and the pellet was discarded. The supernatant

was centrifuged for 20 minutes at 10,000 x g to obtain the

mitochondrial pellet and postmitochondrial, soluble

fraction.

Extraction of Enz mes. The post-mitochond.rial, soluble

fraction was brought to 60% w/v! with solid NH !�SO

protein precipitate was dissolved in 0.01M Tris, pH 8.0,

and dialyzed overnight against the same buffer. This prep-

aration contained OTC cytoplasmic!.C

Mitochondria in the pellet were washed with extracting

buffer and resuspended in 0.01M Tris, pH 8.0 for 1 hour.

During this period the mitochondria were permitted to

break, and debris was subsequently removed by centrifuga-

tion �5,000 x g for 5 minutes!. Protein in the super-

natant was precipitated, resolubilized, and dialyzed as

described for the soluble fraction. This preparation con-

tained OTC mitochondrial!.

Determination of OTC. In the direction of citrulline

synthesis, OTC was assayed essentially by the method of

Ong and Jackson 81!, using 1.0 ml of 0.1M Tris pH 8.0!,

5mM ornithine, and freshly prepared 5 mM carbamoylphosphate.

Enzyme preparations were added to produce between 30 and

100 nmol of citrulline during a 10 minute incubation period

37C. Citrulline was determined according to Prescott

and Jones, Method II 85!.

29

OTC in the direction of ornithine formation was assayed

by the radioisotope procedure of Reichard 87! which meas-

14 14ures CO released from C-ureido labeled citrulline New

2

England Nuclear! in the presence of arsenate and enzyme.

Unlabeled citrulline was added as shown in the results, and

0.5ml of extract was used with 1.0ml of the reaction mix-

ture, containing 0.1M citrate buffer pH 7.0! and 50mM

arsenate. Incubations in sidearmed flasks were for 1 hour

at 30C, and the reaction was terminated by the addition of

0.5ml of 6N HCl and 15% trichloroacetic acid from the side-

arm. Radioactivity was measured in a liquid scintillation

counter in scintillation fluid containing PPO, POPOP, and

toluene. Thin-layer chromatography of the assay mixture

after incubation showed unreacted citrulline as the only

radioactive substance.

Determination of Succinic Deh dro enase SDH!. SDH was

assayed according to the method described by Veeger et al.

�41!. Controls were included to correct for interfering

oxidase reactions in the cytoplasmic extracts. No de-

pression of SDH activity by cytoplasmic extracts was

observed.

Determination of Glucose-6-phosphate Isomerase GPI!.

To eliminate sucrose interference in the assay procedure

for GPI, 0.5M mannitol instead of sucrose was used in the

extraction medium for mitochondria. GPI was assayed in

mitochondrial and post-mitochondrial extracts by the

30

procedure of Reithel 90!.

Molecular Wei ht Estimation. OTC and OTC were

chromatographed separately on a Sephadex G-200 column �.5

x 44.5cm! at a flow rate of 0.1 ml/min; V = 213ml, Vc 0

55ml. The column was calibrated with proteins of known

molecular weight peroxidase, alkaline phosphatase, beta-

amylase, and urease! which were identified by relative peak

heights A ! and by enzymatic activity.

Protein Determination. Protein was measured by the

method of Lowry et al. �7!.

Results

Intracellular Distribution of OTC. In one preparation,

total OTC activity in the post-mitochondrial, soluble frac-

tion was 10 times greater than in the mitochondrial frac-

tion Table I!, but, expressed in terms of protein, the

enzyme designated as OTC had greater specific activity.

Comparison with SDH indicated that OTC was associated with

a fraction that also contained most of the mitochond.rial

The distributions of OTC and OTC were compared with GPIC H

as well as SDH by percent distribution Table II! . Con-

tamination of the post-mitochondrial fraction by broken

mitochondria averaged 27%, as shown by SDH activity. How-

ever, post-mitochondrial contamination of mitochondria was

very low, since only 4% of GPI activity occurred in the

mitochondrial fraction. Total OTC activity was 75% in the

31

post-mitochondrial fraction and 25K in the mitochondria,

and this distribution is significantly different from

either marker enzyme.

Subcellular distribution of OTC's was investigated in

extracts from the mitochondrial pellet and from the post-

mitochondrial fraction of sugarcane cells. OTC's in these

extracts had different retention characteristics on a DEAE

cellulose column Figure 6!. OTC from the principle peakN

of the mitochondrial fraction eluted when a concentration

of approximately 0.47N phosphate was reached, whereas OTC

eluted with a much lower phosphate concentration �.15N!.

When the two previously separated fractions were recom-

bined and the column was charged with a 1.75:1 ratio of

OTC to OTC , the enzymes were recovered with a ratio of

2.88:1 Figure 7!. Thus, a larger proportion of the mito-

chondrial than the post-mitochondrial enzyme appears to be

lost by the DEAE cellulose column procedure. The loss

could have resulted from inhibition of this enzyme by phos-

phate in the eluting buffer 8!.

When OTC and OTC were applied to agarose gel column

in a ratio of 0.33:1, they were recovered in approximately

this ratio Figure 8!. The larger, OTC , peak eluted aheadM 7

of the smaller, OTC , peak. Since agarose gel is a molecu-C'

lar sieve, the experiment indicated a higher molecular

weight of OTC than OTCN C

Relative Molecular Size. An approximation of molecular

weights of the two OTC's was obtained on Sephadex G-200 by

32

comparing their displacement from a column with that of

several enzymes of known molecular weights see Naterials

and Methods!. On this basis molecular weights of OTCC and

OTC were calculated as 79,000 and 224,000, respectively

Figure 9!.

Kinetic Pro erties. In the forward reaction, and with

an ornithine concentration of 10mN, OTC had a sharp peak

of activity at pH 7.5, while OTC activity had a broader

optimum pH, ranging from pH 7.5 to 8.5 Figure 10!. In the

reverse reaction, the activity of both enzymes peaked

sharply at pH 7.0 Figure 11!.

The effect of pH on the forward reaction citrulline

formation! depended on the ornithine concentration in the

incubation mixture. When pH was kept constant, there was

appreciable substrate inhibition, particularly of OTC

Figure 12!. At pH 8.5, maximum activity for both OTC andC

OTC was reached at about lmM ornithine. At higher concen-M

trations, there was, a rapid decrease in activity ~ At pH 8,

the pattern for OTC was similar, although substrate in-

hibition was less severe. At this pH, OTC had a much

broader activity spectrum: maximum activity occurred be-

tween 3 and 4mM ornithi ne, and inhibition by ornithine was

only 23X, even at a substrate concentration of 10 mM.

When ornithine was added at concentrations below that

needed to produce inhibition, the activity of both OTC's

increased up to pH 9.0, the highest pH tested Table III!.

33

On the other hand, when ornithine concentrations were ad-

justed to yield a constant concentration of the zwitterion

form of pK . 8.65! at each pH, OTC activity remained rela-

tively constant Table IV!,

At pH 8.0 and a saturation concentration of carbamoyl-

phosphate, Michaelis-Mentan kinetics prevailed for OTCC and

a K value of 3.11mN was calculated Figure 13a!. OTC had

a much higher affinity for ornithine K = 0.5mB! Figurem

13b!. The Lineweaver-Burk plots indicated that high orni-

thine concentrations inhibit OTC more than OTC - No in-

hibition was observed when K values for carbamoylphosphatem

were determined in a similar manner and no appreciable

differences of the K values for carbamoylphosphate werem

found between the two enzymes OTC = 0.12mM; OTC O.llmM!C

Figure 14a,b!.

Contrary to findings for the forward reaction, the for-

mation of ornithine from citrulline required not only a

higher concentration of substrate for saturation, but OTC

had a higher affinity for citrulline than did OTC Figure

15a,b!.

Inhibition by Ar in.ine. Previously �4!, it was demon-

strated that cultures of aging sugarcane cells grown in the

absence of exogenaus arginine divide less rapidly than simi-

lar cells grown in media containing arginine. The specific

activities of the two OTC's in cells grown far 6 ta 8 days

the. presence and absence of exogenous arginine were

34

compared Table V! . The activity of QTC increased betweenM

two and three-fold in the absence of exogenous arginine,

while OTCC did not respond to arginine supplementation; its

activity, under both conditions, remained comparable to

that of OTC in the arginine-supplemented medium. ResultsM

were variable but statistically significant.

In vitro, arginine did not inhibit OTC or OTC fromM C

sugarcane cells, even when arginine exceeded by a factor of

10 the maximum concentration at which it is likely to be

present in the cells �06!.

Discussion

Existence of two OTC's in sugarcane cell suspensions

corroborates reports by others that higher plants have mul-

tiple forms of this enzyme �6,118!. The two OTC forms

differ in their relative mobilition on gel filtration,

their intracellular locations, and their kinetic properties

with respect to ornithine and citrulline. Cell levels of

OTC but not OTC are decreased in the presence of exogen-M C

ous arginine. The two forms are similar in their affini-

ties for carbamoylphosphate in the forward reaction. The

elution pattern of sugarcane OTC's from a DEAE cellulose

column resembled the elution pattern from a similar column

reported for pea seedlings �6!. Multiple OTC's from pea

seedlings �6! and apple leaf tissue �18! have been dis-

tinguished on the basis of their pH optima in the forward

35

reaction. However, in sugarcane, OTC's do not differ

fundamentally in their pH response. For both forms, as for

human liver OTC, pH appears to affect activity largely by

the concentration of ornithine zwitterions present and

these may be the true substrate at both catalytic and in-

hibitor binding sites l17!. Differences in pH optima are

related to differences in K and K. values for ornithinem i

and are dependent on the concen.tration of ornithine in the

incubation mixture

Molecular weights calculated from a molecular sieve

column are approximations, but OTC could be a trimer of

the molecular form of the cytoplasmic sugarcane. enzyme.

Similar relationships exist in the molecular weights of

OTC's of Streptococcus faecalis and bovine liver �6!, en-

zymes normally having stable species of 223,000 and 108,000,

respectively. In 6N guanidine-HC1, both of these enzymes

form a monomeric species of approximately 38,000 molecular

weight, and the S, faecalis enzyme forms a stable dimer

i.e. 74,000 mol wt! at pH 9.5 in 0.2M NH4Cl, a species

simi lar to sugarcane OTC, as we11 as to Nostoc muscorum

OTC 8!, Marshall and Cohen �6! propose that the enzyme

is further stabilized by a hexamer configuration i.e,

224,000 mol wt! of the basic unit, but that the dimer also

has functional significance.

The data obtained in these present experiments suggests

that the high molecular weight OTC , isolated by a methodM'

36

yielding chiefly mitochondria, is ni oc on ria, is, in fact, a mitochondrial

enzyme. The smaller enzyme, OTC obo tained from the super-

natant of a 10,000 g centrifugation is 1'k 1i e y to be

cytoplasmic.

Functionally, the two OTC's mas may differ. The enzyme in

the particulate fraction a ec ion appears to favor an anabolic re-

action, while kineti'ne ics for the soluble OTC indicate a cata-

bolic role fe for this enzyme. A hp ysical separation of two

functionally different OTC'ss offers advantages in econo-

mizing cell enerergy and in metabolic regulation

In the direction of oro ornithine synthesis, the affinit

of OTC for citrulline is hi her ts ig er than the affinity of OTC

for this substrate.

M

In the d zrectl.on of citrulline forma-

tion, on the other hand, the sie s tuation is reversed: OTCH

has a higher affinit fory or ornithine than does OTC

F urther evidence for the rela

C

or t e relative inefficiency of OTC asC

an anabolic c enzyme is sub sis su strate ornithine! inhibition

which be ing ns to immit activit weli y we 1 before the theoretical

K fm

or ornithine is attained, whereas OTC is inhibited by

ornithine only when thw en the saturation limit of ho t e enzyme-

approached. The relation h'ions ips between

Pseudomonas OTC's and than t eir respective

i.ffer from m similar relationships for

th hat is associated with an anabolic function, and the

larger which has a catabolic function �50! ' in sugarcane

substrate complex is

molecular weights of

metabolic functions d

sugarcane cell OTC's: in Pseudom omonas it is the smaller OTC

the reverse situation appears to occur.

OTC's in microbial species are under different. regula-

tory controls with respect to arginine �02,150!. In

sugarcane, OTC , the anabolic enzyme is least abundant when

the cells are grown in a medium containing arginine. There-

fore, it resembles the anabolic enzyme in Pseudomonas which

also has a similar pH optimus at 8 to 8.5 �24!. OTC with

an anabolic function is more likely to be sub]ect to re-

pression by exogenous arginine than is the catabolic OTC

i.e. OTC !. Neither OTC nor OTC appears to be inhibitedC

by an allosteric mechanism because exogenous arginine af-

fect.s only cells incubated in the presence of arginine for

several days.

An anabolic function for OTC is in keeping with the

mitochondrial location of OTC in fungi �49! and mammals

�1! and also with observations that isolated mung bean

14 14mitochondria converted C-ornithine to C-citrulline

�0! ~ A catabolic function for OTC is only speculativeC

from kinetic evidence. It must be shown that the reverse

reaction occurs in the chemical environment of the intact

ceil. This possibility is explored in the following

chapter.

38

CHAPTER III

Decarbamoylation of Citrulline

by Sugarcane Cells

39

'roduction

«bamoylases

of citrulline in vitro but so far only certain

facies oof bacteria and algae have been found to decarba-

.late citrulline in vivo �30, 150! . In these organisms

''.j.thine f ollowed by conversion of ornithine to citrulline

j,40,43! . In the present experiments with sugarcane

:gs a direct. assay f or citru11ine decarbamoylation was

"e~'d. on the production o f carbon dioxide f rom the ureido

up of citrulline 87! . Ureido- C-citrulline, f ed to14

14arcane cells in acidic medium, liberated CO which was

'"! ected and measured. Indirect production o f carbon di-

''d;e from the ureido group was minimized by utilizing

'1's in the stat i onary phase of growth. These cells are

';cient in the ability to convert citrulline to arginineI

!.so production of carbon dioxide by the arginase � ure�

~pathway was prevented. Carbon dioxide can also arise

'the decarbamoylation of N-carbamoylputrescine, which is

".dodeca«boxylation product of citrulline. However, sugar-

cells of the variety used here do not appreciably me�

Plize N-carbamoylputrescine whether it arises endoge�

or is supplied in the medium �5!. The possible

~arbamoylation

j-,nine and the

' i,thine 99! ~

e �interpreted

has been assayed by the disappearance of

sequent ial produc t ion o f citrulline and

However, similar results in higher plants

to be due to degradation of arginine to

40

occurrence of either indirect pathway was assessed by ex-

14 14amining cells fed ureido- C-citrulline for C-arginine or

14C-N-carbamoylputrescine.

Naterials and Nethods

Cell-culturin . Sugarcane cells Variety H50-7209! in

suspension culture were grown in a stock medium containing

yeast extract supplemented with 300 pN arginine �6! .

Cells in the stationary phase of growth were used in the

experiments.

Measurement of Citrulline Decarbamo lation by Whole

Cells. Citrulline decazbamoylation was measured by release

14 14of CO from ureido- C-citrulline. In a continuous feed-2

ing experiment, l. 5 gfw cells were placed in Warburg flasks

containing 3 mN citrulline �. 75 mCi ureido- C-citrulline!.14

Cells were incubated at 30oC on a rotary shaker �60 rpm/14

min!. Uptake of isotope into cells and CO production2

were measured at one hour intervals over the course of the

five hour incubation period. A control flask contained

14 C-citrulline and medium from which the cells had been re-

14moved by filtration. CO was trapped in a center-well2

containing 0. 3 ml Hydroxide of Hyamine benzethonium hy-

droxide! and counted in a Beckman LS-150 scintillation

counter, using PPO, POPOP, and toluene. Radioactivity of

cells was measured by drying 0.1 gfw samples of washed

«lls for 3 hours at 110oC prior to counting as described

41

above. In a pulse-label experiment 1.5 gfw were incubated

with isotope as in the continuous feeding experiment

After one hour cells were harvested by filtration, washed

three times with distilled water, and resuspended in 5 ml

fresh medium containing 3 mM unlabelled citrulline. C0214

production was measured at one-half hour intervals over the

subsequent three hour incubation period.

Preparation of Ethanol Extracts from Cells. Cells that

14had been incubated for 5 hours with C-citrulline were

water

Chromato raph of Ethanol Extracts. The two fractions

eluting with 1 N NH and. 2 N NH , respectively, were

chromatographed separately on unactivated silica-gel plates

using propanol:ammonia �:3! and phenol saturated with

water as solvent systems. Plates were cut into 1 cm sec-

tions and scraped into scintillation vials for measurement

of radioactivity. Standard amino acids were detected with

ninhydrin spray. The

exchange column with

fraction that eluted from the cation

H 0 contained less than 1% of total

radioact ivity and was not chroma tographed.

washed three times with distilled water and then extracted

three times in boiling 80% ethanol. The combined extract

was reduced to dryness, dissolved in water, and placed on

+a short column of Dowex-50W-X8 H form!. Fractions e-

luting with H 0, 1 N NH , and 2 N NH were collected, evap-

orated to dryness, and brought to 5 ml with distilled

42

'Pre 'aration of OTC OTC and OTC .were pre-

pared from mitochondrial and post-mitochondrial cell frac-

tions obtained from stationary phase cells as described in

Chapter II.

Assay of OTC 'in the Forward 'arid Revers'e Reactions. The

14citrulline, 0. 2+Ci ureido- C-citrulline, 50 mM arsenate,

Q. 1 N imidazole buf f er pH 7. 0!, and 0. 06 ml OTC or OTCM

�,08-0.15 mg protein!. Tubes were incubated for 3 hours

at 30oC and the reaction was terminated by adding 10 microl

100! trichloracetic acid, which also served to acidify the

14medium to release CO . Control tubes contained the com-

piete reaction mixture with boiled enzyme.

Measurement of Protein. Protein was measured by the

method of Lowry et al �7!.

Results

In a continuous feeding experiment, citrulline accumu-

lated in cells; over the first three hours of incubation,

uptake was approximately linear with time Figure 16a!.

the same period cartton dioxid.e was produced from

citrulline Figure 16b! at a rate of 0.46 micromole/ hour/

assay for OTC in the direction of ci,trulline synthesis was

as described in Chapter II. The assay for OTC in the

direction of ornithine synthesis depended on the disappear-

14ance of C-citrulline from the assay mixture. The incu-

bation mixture contained total volume 0.1 ml!: 3mM

43

gfw calculated from data in Figure 16b!. The calculated

rate assumes that negligible amounts of citrulline were

initially present in cells, Citrullin.e was not decarba-

moylated by the spent medium Figure 16c!.14

C-citrulline was the only labelled compound found in

ethanol extracts prepared from cells incubated for five

hours with the isotope Table VI!. Lees than 5% of label

was in the ethanol-insoluble fraction i.e. protein!.

Decarbamoylation, also, was demonstrated in cells that

were pulse-labelled with C-citrulline Figure 17!. CO14 142

evolution peaked one hour after cells were removed from la-

belled medium Figure 18! which may represent the equili-

14bration. period for CO to saturate the medium pH 5.6!

14and enter the gas phase. The decrease in CO production2

observed thereafter may have been. caused by an isotope

diluti,on effect as unlabelled citrulline entered the cells

14About 5% of the C-citrulline taken up durin.g the pulse

period was decarbamoylated during the 3 hour post-pulse

period.

OTC and OTC were extracted from cells in the station-N

ary phase the same stage of growth as those used in the

above experiments! and their activities in citrulline and

ornithine synthesis were measured Table VII!. OTC cata-C

lyzed the decarbamoylation of citrulline at five times the

rate of OTC , based on total activity units. The amount of

present could account f or an in vivo rate o fC

decarbamoylation of 0. 24 micromole/ hour/ gfw, which is

half the observed rate in vivo. The amount of citrulline

supplied in the enzyme assays � mM! was well below the Km

of either enzyme for this substrate Chapter II! but it

probably exceeded the concentration of citrulline in the

cells.

Discussion

14Ureido- C-citrulline was infiltrated into apple stem

14tissue �6! in a similar experiment. Production of C02

was reported in a data table, but was not commented on; in-

stead, the role of citrulline in arginine synthesis was

discussed. In the present experiment, arginine synthesis

accounted for little, if any, of citrulline metabolism.

The principle reaction was decarbamoylation. This almost

14certainly occurred directly, because neither C-arginine

14nor C-N-carbamoylputrescine was detected in cells fed

14C-citrulline for five hours.

If it is assumed that half the activity was lost on ex-

traction sufficient OTC was present in cell-free extractsC

to account for the in viva rate of decarbamoylation ~ On

the other hand, 90% of OTC would have to be lost on ex-

traction for this enzyme to be accountable.

Catabolic OTC's are usually found in bacteria that con-

«in the arginine desiminase pathway. The following two

chapters describe attempts to demonstrate arginine

45

desimi nase, in cell-f ree extracts, and whole cells, re-

spectively. In the process, other aspects of arginine

catabolism are also examined.

46

CHAPTER IV

Enzymes of Arginine Catabolism in Sugarcane Cells

Introduction

Citrulline can arise from the action of arginine de-

siminase on arginine. In turn, citrulline is conceivably

an intermediate in the production of glutamic acid and N-

carbamoylputrescine, both products of arginine catabolism

in sugarcane cells �5!. On the other hand, arginine in

higher plants usually gives rise to glutamic acid by the

arginase pathway �20! and to N-carbamoylputrescine by the

agmatine pathway �14!, neither of which involves citrul-

line as an intermediate.

desiminase, arginase, urease, and arginine decarboxylase.

Materials and Methods

Pre aration of cell-free extracts and rotein

concentrates. Sugarcane cells were cultured as previously

described Chapter II!. Cells in the linear and station-

ary phase were harvested and washed. Cell � free extracts

were prepared by grinding 1 to 5 gfw cells in one volume

ice-cold buffer �.1M Tris, pH 8.0; or 0.1M sodium phos-

phate, pH 6.5! in a chilled mortar with pestle. The brei

was squeezed through 4 layers of cheesecloth and then

centrifuged 10 minutes at 15,000 xg to remove cell debris

These experiments attempted to resolve the question of

whether citrulline is an intermediate in arginine cata-

bolism in sugarcane cells by testing for the existence of

the following enzymes in cell-free extracts: arginine

48

The clear supernatant was designated the .cell-free extract

and served as the enzyme source in some assays. In others,

a protein concentrate was prepared by precipati;ng pro-

tein by the slow addition of solid NH ! SQ to 60% w/v!.4 2

The protein was recovered by centrifugation and then re-

dissolved and dialyzed overnight against O.OIM extracting

buffer. In same experiments, mitochandrial and cytoplas-

mic fractions were prepared as previously described Chap-

ter II!. Protein concentrates of the fractions were pre-

pared as above.

Determination of ar inine desiminase. The assay for

14arginine desiminase was based on the production of C-

14citrulline from guanidino- C-arginine. A typical incuba-

tion mixture contained in a total volume of 0.2ml: 0.18ml

l4enzyme; 10 ~ guanidino- C-arginine I Ci!; and 10 pl L-

arginine �0 mN!. Control tubes contained boiled enzyme.

Tubes were incubated. for one or two hours at 30 C and the

reaction was stopped by heating tubes in a boiling water14 l4

bath for 5 minutes. G-citrulline was separated from C-

arginine by paper chromatography. 10 micraliter aliquots

of incubation mixtures were spotted onto Whatman //1 paper,

Chromatograms were developed in a descending system in

which solvent was allowed to drip from the end of the paper

for several hours in order to increase the separation of

citrulline and arginine. The solvent system was n-butanol:

acetone: diethylamine: H 0 �0;70;14;35! and typical rf2

49

values. were; arginine,, 07; citrulline> -. 28; orni,thine,

. 35; urea, . 47. Radioactive spo ts were located by radio-

autography, and radioactivity was measured by cutting out

spots and counting in a liquid scintillation counter in

Actuasol acintillatiun fluid New England Nuclear!. Aminu

14 14duced from guanidino- C-arginine is converted to CO by14 CO is trapped in a center well

2adding urease Sigma!

containing Hydroxide of Hyamine. Incubation mixtures con-

tained in a total volume of 0.5ml; O.lml enzyme; 0.4ml140.1M L-arginine pH 9.7!; and 8pCi guanidino- C-arginine.

The enzyme source was dialyzed protein concentrate, taken

up in 0.01M Tris buffer, pH 8.0. In. one assay the enzyme

was activated by heating it for 20 minutes at 55oC in the

in other assays the activationpresence of 0.01M MnC12,

step was omitted. Plasks were incubated for 30 minutes at

30oC.

Determination of Urease. Urease was assayed by trap-

14 14ping CO produced from C-urea. An incubation mixture2

contained in a total volume of 5ml: 1.0ml enzyme; 4ml .1M

sodium phosphate buf f er pH 6. 0! with 3mM urea; and 0. 5

pCi C-urea. The incubation period was one hour, at14

1430 C. CO was trapped in Hydroxide of Hyamine and2

counted in a liquid scintillation counter in a liquid

acids standards were located by spraying with ninhydrin.

Determination of ar i'nase. Arginase was assayed by14

the micro-procedure of Schimke 98!, in which C-urea pro-

50

Results

scintillation fluid containing PPQ, POPOP, and toluene.

The enzyme source was protein concentrate taken up in .1M

sodium phosphate buffer pH 6.0!.

Determination of ar inine decarbax lase. Arginine de-

14carboxylase was assayed by the production of CO from

14uniformly labelled- C-arginine. The incubation mixture

contained in a total volume of 5ml: 1.0ml enzyme; 4ml so-

dium phosphate buffer pH 6.0! with 3mM L-arginine; and 0..5

pCi uniformly-labelled � C-arginine �16!. The incubation14

period was 1 hour, at 30oC. CO production was measured14

14as for urease. Production of CO from the guanidino

2

carbon was very low compared to that evolved from all six

carbons see Results!. This supports the specificity of

the assay f or arginine decarboxylase since only the 1 � carbon

can logically be split without the prior removal of the

guanidino carbon.

No arginine desiminase activity was detected in ten

separate enzyme preparations. In some experiments cell-

free extracts were used and in others dialyzed protein

concentrates were used. Assays were conducted both at pH

8.0, which is the pH optimum for the enzyme from Chlorella

�03!, and at pH 6.5, which is the optimum for bacterial

arginine desiminase 98!. The cells used as starting ma-

terial varied in age fram 1 week linear growth stage! to

IIII&'tIII e,", Wi

sa

", Ilail

51

weeks stationery stage! . In one experiment C-arginine14

was added carrier-free.

A problem with the assays was that the stock C-argi-14

14nine already contained 5 to 8% C-citrulline as a contami-

nant, which reduced the sensitivity of the assay. The

stock isotope was finally repurified twice by chroma-

tography on the standard system! to contain less than 0.1%

citrulline. But, still no arginine desiminase activity was

detected Table VIII!.

Arginase, also, appeared to be absent from the extracts.

It would have been detected in the above experiments at pH

148.0! by the production of C-urea, or by the loss of

14counts in C-arginine, assuming that urea is rapidly de-

graded by an endogenous urease. But no C-urea was de-14

tected on chromatograms, and little if any C-arginine was14

14metabolized to C02 Table VI II! . A specif ic assay f or

arginase was conducted at pH 9.7 using a micro-assay, with

negative results.

Arginine decarboxylase and urease were both present in

protein concentrates Table IX!. Interestingly, the two

enzymes appear to exist in different cell compartments;

urease was chiefly in the post mitochondrial fraction while

arginine decarboxylase was almost all in the mitochondrial

fraction. Urease activity was 2.6 nmol/ min/ gfw cells;

arginine decarboxylase activity was an order of magnitude

higher, 27 nmol/ min/ gfw cells rates calculated from data

52

shown in Table IX! .

Discussion

Arginine desiminase has not been found in any higher

plant, although it is present in bacteria 98! and algae

�03,135! ~ Arginase has been found in a number of higher

plants �3,120!, but it was absent. from tomato and cactus

�0!. Neither enzyme was. found in sugarcane cells. The

question of how glutamic acid is produced from arginine in

these cells was not answered, since the two known biochemi-

cal routes start with either arginine desiminase or argi-

It is possible that the enzymes were inactivatednase.

volvement of the agamatine pathway in the production of N-

carbamylputrescine from arginine. The enzyme has been

found in other higher plants that contain the agmatine

pathway. In contrast to barley, in which the enzyme is in

during the extraction procedure; but, neither enzyme is re-

ported to be especially labile in vitro. It may be that

sugarcane cells metabolize arginine to glutamic acid by

some other route.

The presence of urease confirms other reports that the

enzyme is common in plant tissues 88!. As in other plants

the physiological role of urease in sugarcane is not clear,

since there has not been found a source of endogenous urea

for it to act upon.

The presence of arginine decarboxylase supports the in-

53

the soluble fraction. �16!, in sugarcane cells it was al-

most all in the mitochondrial pellet.

From these experiments there is no evidence that

citrulline is an i.ntermediate in arginine catabolism in

sugarcane, This possibility is further explored in experi-

ments with whole cells, described in the next chapter.

54

CHAPTER V

Production of Carbon Dioxide from Arginine and Urea

by Sugarcane Cell Cultures

Introduction

Most plants have the ability to catabolize arginine.

The carbon skeleton is often largely degraded to carbon

dioxide, while 99% of the nitrogen is retained by the plant

�22,138!. The six carbon atoms produce carbon dioxide

potentially by four pathways. The guanidine carbon may be

respired in either the arginase pathway with urea as an

intermediate! or the arginine desiminase pathway with

citrulline as an intermediate!. The 1-carbon can be lost

in the agmatine pathway, which in sugarcane cells gives

rise to N-carbamoylputrescine, which is not further meta-

bolized �5!. Carbons 1 to 5 can also give rise to carbon

dioxide in the citric acid cycle, via ornithine which en-

ters the cycle by its prior conversion to alpha-keto-

glutarate,

The present experiments examined arginine catabolism

in sugarcane cells with the primary goal of identifying

either the arginase pathway or the arginine desiminase

14 14pathway, based on the production of either C-urea or C-

14citrulline from guanidino- C-arginine. Production of

14 14CO from C-urea was also measured and the contribu-

2

tion of carbons 1 to 5 of arginine, to total arginine res-

piration, was compared to the. contribution from the

guanidino carbon alone.

Naterials and Nethods

A11 of the methods have been described in preceding

chapters, or variations are indicated in legends to figures

or in the text. Isotope feeding experiments were conducted

exactly as described in Chapter III except. for the differ-

ent isotopes used.

Results

14Guanidino- C-arginine was fed to cells in the presence

of 3mN unlabelled arginine. Uptake of isotope into cells

14and production of CO were roughly linear over the first

three hours Figure 19! and in this period carbon dioxide

was produced at a rate of 0.144 ~ol/ hour/ gfw cells cal-

culated from data in Figure 19!.

hourly intervals, cells from an entire flask were

washed and extracted three times in boiling 80% ethanol.

The extracts were chromatographed on unactivated silica-gel

TLC plates with propanol: H 0 �:1! as the solvent system.2

Regions corresponding to arginine rf=.04! and citrulline

r f=. 66! were cut out and measured. f or radioactivity. From

this experiment it appeared that about 20% of the label was

present in citrulline after a one hour incubation Table

X!. These results, however, were not reproducible in two

subsequent. experiments, in which different solvent systems

were used for chromatography. When descending paper

chromatography with n-butanol: acetone: diethylamine: H 02

57

�0:70:14:35! as a solvent system was used, very few counts

were found in either citrulline or urea, in a sample incu-

bated for one hour with isotope Table XI!. Similar re-

sults were obtained with samples incubated for one-half,

two, and three hours with isotope. At least two unidenti-

fied radioactive products were found on these chromato-

grams, which could account for the radioactivity attributed

to citrulline in the first experiment.

An experiment was conducted to determine if whole cells

could respire urea, and also to compare the contribution of

carbons 1 to 5 to that of the guanidino carbon in carbon

dioxide production from arginine. Cells were incubated for

three hours in media containing 3mM urea with 384,515 cpm

14C-urea!, or 3mM L-arginine with 711,060 cpm uniformly-

labelled arginine!, or 2mM L-arginine with 831,515 cpm

14guanidino-labelled arginine!. Production of CO was2

measured for each treatment and the results were expressed

as rates of carbon dioxide production Table XII!. Urea

supported the highest rate of carbon dioxide production

but, the cells were damaged by the treatment they were

brown and clumped after three hours!. Production of car-

bon dioxide, from all six carbon atoms of arginine, was

calculated from the results with uniformly-labelled argi-

nine to equal 0.162 pmo1./ hour/ gfw cells. Respiration of

the guanidino carbon alone was calculated to equal 64% of

the total.

58

Discussion

Under the experimental conditions used here the release

of carbon dioxide from the guanidino group of arginine was

quantitatively more important than it. was from carbons 1 to

5 combined. Carbon dioxide that was not derived from the

guanidino group may have come mostly from the l-carbon, in

view of the high arginine decarboxylase activity of these

cells Chapter IV!. It has been shown previously �5! of

sugarcane cells that uniformly-labelled arginine gives rise

to the glutamic acid family of products, so it is logical

to conclude that release of carbon dioxide from the guani-

dino group is a measure of glutamic acid production.

Respiration of the guanidino carbon of arginine was

some three-fold slower than either the decarbamoylation of

citrulline Chapter III! or the respiration of urea. Thus,

either compound could theoretically be an intermediate in

arginine respiration, with no detectable accumulation of

14either compound in the cells. In fact, neither C-citrul-

14line nor C-urea was positively identified in cells fed

14guanidino � C-arginine. Therefore, neither these nor pre-

vious experiments support a role for citrulline in arginine

catabolism in sugarcane.

59

CHAP 7 ER V I

Conclusion

The hypothesis that OTC is an anabolic enzyme is sup-N

ported by its partial repression by arginine, which is a

common feature of bacterial �4,86! and fungal �1! ana-

bolic OTC's. Also, OTC is more efficient in ci trulline

synthesis than OTC , judging from its higher affinityC t

lower Km! for ornithine.

The hypothesis that OTC is a catabolic enzyme is par-G

tially supported by evidence. Three criteria were tested

to shaw that OTC acts primarily in citrulline decarba-C

moylation in vivo: kinetic specialization of the enzyme

for the reverse reaction; decarbamoylation of citrulline by

whole cells; and the existence of the arginine desiminase

pathway in the cells. Only the first two criteria were

met.

Kinetic specialization is evident from the five-fold

higher affinity af OTC for .citrullin.e, compared to OTC<.

However, the enzyme is,nat as specialized as the catabolic

OTC from Pseudomanas flourescens �50!, which is subject

to feed-back inhibition by putrescine, the catabolic end

product of arginine in that organism. Na comparable regu-

latory property was found for OTC

Whole cells decarbamoylated citrulline at a rate most

easily explained by the action of OTC . Neither arginineC

nor N-carbamoyl-putrescine appeared ta be intermediates in

the process, nor was sufficient OTC recovered from theM

cells to account for the in vivo reaction rate

61

On the other hand, arginine desiminase activity was not

detected, either in cell-free extracts or whole cells, and

no role was found for citrulline in arginine catabolism.

Naretzki et al. �5! proposed that citrulline may be an

intermediate in the production of N-carbamoylputrescine in

sugarcane cells; however, the presence of arginine decar-

boxylase argues instead for the agmatine pathway. The

presence of urease shows that citrulline is not necessarily

an intermediate in the production of glutamic acid from

arginine, although the key enzyme arginase was not de-

tected, More experiments are needed to determine the path-

ways of arginine catabolism in sugarcane cells. Unless an

endogenous source of catabolic citrulline is found, a cata-

bolic role for OTC does not seem likely.C

Multiple forms of OTC have been found in other plants

�6,118!, and activity has been found in both mitochandrial

�0! and cytoplasmic �9! cell fractions. The experiments

with sugarcane support a unifying hypothesis: separate

mitochondrial and cytoplasmic forms of OTC exist in higher

plants. Multiple forms of OTC are common in bacteria �2!,

54,86,150!, and not in every case is there an obvious func-

tional specialization of the different farms �2,54!. In

contrast, mammals �9! and fungi �1! have a single mito-

chondrial! OTC. A conclusion of the literature review was

that higher plants are closer to bacteria than mammals in

their arginine metabolism. The conclusion is supported by

62

the evidence with respect to OTC's in sugarcane cells, al-

though the sugarcane OTC's are differentiated from bacteri-

al enzymes in their subcellular compartmentation.

63

TAELZ5

Table I. Distribution of

Orni thine Transcarbamoylase Activityin Subcellular Fractions

Post-mito-

chondrial

fraction

Mito-

chondrial

fraction

Ornithine

transcarbamoylaseSpecific activityTotal2

40. 2

126.6

100, 0

12. 0

Succinic

dehydrogenaseSpecific activityTotal activity

0.06

0.20

6.00

0.72

Total protein mg! 0.12 3.15

1nmol citrulline formed/min g fresh wt cells

2nmol citrulline/min g fresh wt cells

3Change in A /min ' mg protein

4Change in A /min g fresh wt cells

Table I I. Dis tr ibut ion of Orni thineTranscarbamoylase and Marker Enzymes

in Subcellular Fractions

Percent activities are based on total amount of each enzyme

Experiment Ornithine Succinic Glucose-6-phosphate*

transcarbamoylase dehydrogenase isomerase

81.1

88 ' 4

81.1

72.0 28.0

81.7

48.3

8.6 87.0

78.0

13.0

22.0

4.0 96. 0

10 89.3 95.94.1

96.988.3

48.1

3.1

12 55 ' 0 45.0

96.325.2 74.8 27. 073.0 3 ~ 7means:

~OTC distribution is significantly different than either markerenzyme at 95X confidence level by Mann-Whitney U-test �1!.

Mito .

18. 9

ll. 6

18. 9

18.3

51.1

31.0

21.0

10.7

11.7

51.9

Post-mito. Mito. Post-mito. Mito ~ Post-mito.

48.9

51.7

91.4

69.0

79.0

66

Table III. Effect of pH on OTC'sat Constant Ornithine Concentration �.5 mM!

in the Direction of Citrulline Synthesis

Activities are expressed as % of activity at pH 9.0.

OTCOTCG

145 nmol/min g fresh wtcells

23. 2 nmol/min ' g fresh wtcells

7.0

7.5

8.0

8.5

9.0

15

26

52

/31001

12

31

63

931002

67

Table IV. Effect of pH on OTC Activitiesat Non-Saturating Ornithine Concentrations

Ornithine concentrations were varied to give equal concen-

trations of alpha-NH species pX 8.65! at each pH value.2

Percentage activities are based on activities of OTC's1

at optimal ornithine concentrations �.0 mM for OTC , and

3.0mM for OTC ! at pH 8.0.2

OTCOrn

1.8 O.D. units/10 min 0.1 mlextract0.643 O.D. units/10 min 0,1 mlextract

7.5

8.0

8.5

9.0

1.40

0.50

0.22

0.13

41

46

64

50

OTC

67

65

65

63

68

Table V. Effect of Exogenous Azginineon Cytoplasmic and Mitochondrial OTC's

in Sugarcane Ce11s

Flasks were inoculated at start of experiments with 1 week-old washed stock cells from a single culture. OTC in thedirection of citrulline synthesis was determined after 6 to8 days' incubation of the cells in a synthetic medium �6!supplemented with 300 .N L-arginine +!, or without argi-nine -!.

OTCC

OTC

Experiment + +

nmol/min ' mg solubleor mitrochondrial protein

SD

10 ' 4 16.1

5 ' 470.58

1Significant difference between means at 95/

confidence interval.

1 2 34

Nean

54

166

44

53

79

28

186

44

35

73

44

134

43

38

65

157

220

86

146

152

Table VI. Di stribution of P adioactivityin Cells 'Fed Ureido-l4C-citrulline

f or Five Koux's

cpm

Compound 1 N NH 2 N NH

citrulline 1616 77

arginine 0

N-carbamoyl-putrescine

0urea

Compounds listed are possible recipients of label. "INNH>" and "2N NE3" refer to elution from. Dowex 5QW-X8 H+form! column see Materials and Methods!. Counts onchromatograms represent 2% of total from the cell extract.

70

Table VII. OTC .and OTC Activitiesin the Direction of CitruljI'ine Forward!

and Ornithine Reverse! Synthesis

Specific activities are based on mg cytoplasmic OTC ! ormitochondrial OTC>! protein. Total activities are kasedon units per gram of cells wet wt.!.

nanomole/ min/ mg micromole/ hr/ gfw21.9

Reverse 1.7

OTC

46.8

2.5

OTC

Forward

Forward

Reverse

SpecificActivity

Total

Activity

3.10

0.24

0.96

0. 05

Table VIII. Apparent Absence of ArginineDesminiase and Arginase from Sugarcane

Celj-free Extract

cpm/ 10 microliters

Sample arginine citrulline urea

Control

Complete /tl

Complete /2

48062

47826

49576

364 51

381 29

302 28

Extract was prepared in 0.1 M Tris, pH 8.0, and assayedimmediately. Control tube contained boiled instead of ac-tive enzyme preparation. In two tubes with active enzymepreparation Complete fi'1, I/2! no radioactivity above thecontrol was found in citrulline or urea, nor was the re-covery of C-arginine less than in control tube. Produc-tion of C-citrulline would indicate the presence of14

arginine desiminase, of C-urea, arginase.

72a

cd

D

b0

I

I I

I

0oCfl

I

cd

0 C~ R b0

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73

Table X. Distribution of Radioactivityin Arginine and Citrulline in Sugarcane Cells

Incubated in Guanidino-14C-arginine

T ilIle

hrs! citrulline

cpmsr inine

C P II!

citrulline

1228 293 19.3

1350 122 8.3

1796 106 5 ' 6

2336 154 6.2

3948 254 6.0

The cells were obtained from the experiment described inFig. 19. At designated times cells were harvested, washedthree times with distilled water, and extracted threetimes in boiling 80% ethanol. Extracts were reduced to asolid an.d brought to 0.5 ml each with distilled water.10 microliter aliquots were chromatographed on unactivatedsilica-gel TLC plates using propanol; H20 �:1! as solventsystem. Regions corresponding to arginine and citrullinewere cut out and counted in a liquid scintillation counter.

Table XI. Distribution of Radioactivityin Stock Guinidino-14 C-arginine and in an

Ethanol Extract from Cells Incubatedfor One Hour with Guanidino-14 C-arginine

Sample C PIll C PIllcpm

arginine citrulline urea

14 .. *stock C-arginine

1 hour sample

14972

49541207

This was an 0.1 microliter aliquot from the stock guani-dino- C-arginine which had been repurified by chroma-14

tography see Results!.

1.5 gfw cells were incubated for 1 hour at 3shaking in 10 ml medium containing 3 mN L-ar106 cpm guanidino � C � arginine!. At end of14

period cells were harvested, washed, and extscribed in Table I, and chromatographed as dthe text. Regions corresponding to arginineand urea were cut out and counted by liquid

OoC with

gxnxne �.6 xincubation

racted as de-

escrxbed xn

citrulline,scx.ntxllatj.on.

75

Table XII, Production of Car'hon Dioxideby Sugarcane Cells from Urea, the Guanidino

Carbon of Arginine �C!, and from allSix Carbons of Arginine �-6C!

14the production of CO from a measured amount of radio-2

active substrate. Substrates were: Flask 1, 3 mN urea

�84,515 cpm!> Flask 2� 3 mN L-arginine 831,515 cpm guani-

14dino � C-arginine!; Flask 3, 3 mM L-arginine �11,060 cpm

uniformly-1abelled- C-arginine!. Flasks contained 1.5 gfw14

cells in 10 ml medium, pH 5.6, and wexe incubated for three

hours at 30oC with shaking. CQ was trapped in center14

wells containing Hydroxide of Hyamine and counted in a

liquid scintillation counter.

Flask CO Production

mole/ hour/ gfw cells!

0.360

0.103

0.162

Carbon Soutce

urea

6C of arginine

1-6C of arginine

In each flask carbon dioxide px'oduction was calculated fxom

76

'FIGURES

LEGENDS TQ F'INURES

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5 ~

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

The. urea cycle in mammals, from �9}.

Model of the ornithine cycle in plants.

The arginine desiminase pathway.

The agmatine pathway, from �14!,

Ornithine biosynthesis in Chlamydomonas �8},

Elution of OTC -0-! and OTC -Ch;! on DEAE-C M

cellulose. Enzymes were eluted by a linear

gradient of 0.05 to 0.5 posassium phosphate

buffer, pH 7.5 from a 2 x 15 cm column at a

flow rate of 0.3 ml/ min. Volumes in the mixing

flask were 400 ml for OTC and 200 ml for OTC

Elution of a mixture of OTC and OTC on a DEAE-

cellulose column identical to that described in

Fig, 1. Volume in the mixing flask was 400 mls

Elution of a mixture of OTC and OTCM on AgaroseC

gel. Enzymes were eiuted by .01 M Tris, pH 8.0

from a 2 x 58 cm column at a flow rate of 0.3

ml/ min. 5 ml fractions were collected and ana-

lyzed for OTC activity.

Molecular weight estimations for OTC and OTC on

Sephadex G-200 gel. See Materials and Methods

for details of experiment.

Effect of pH on the forward OTC reaction for OTC

N ! and OTC +-! with 10 mM ornithine in the

78

incubation mixture. Contxol rate �QQ%.! f' or QTCC

was 21.9 nmoles/ min/ mg protein and for QTCM

46. 8 nmoles/ min/ mg protein

11. Effect of pH on the reverse QYC reaction for QTCC

Fig.

-C7-! and OTC -4;! citrulline concentration wasM

.1M; arsenate, .05M.

12. Effect of ornithine concentration on OTC -0-C

� ~ -! and OTC Q-, -k-! in the direction of

citrulline formation at pH 8.0 -h. � , -0-! and pH

8.5 -k-, � ~ -!. Control rate �00%! for OTC wasC

33 nmoles/ min/ mg protein and for QTC was 64M

nmoles/ min/ mg protein.

13. Double reciprocal plots showing saturation of QTCC

Fight

Fig.

thine concentration was 5 nN.

15. Double reciprocal plots showing saturation of OTCC

a! and QTC b! by citrulline. Arsenate concen-M

tration was .05M, pH was 7.0.

1416. Decarbamoylation of ureido- C-citrulline by

sugarcane cells in a continuous feeding experiment.

A shows accumulation of label in cells; B shows

Fig,

Fig.

release of CQ from cells; C shows release of14

a! and OTC b! by ornithine. Carbamoylphosphate

concentration was 5 nM and the pH was 8.0. Lines

were fitted by the least squares method.

Fig. 14. Double reciprocal plots of OTC a! and OTC b!C M

showing saturation by carbamoylphosphate. Orni�

79

14 CO from a contxol flask containing medium fromwhich cells were removed by filtration Whatman

14tive production of CO during the 3 hour post-2

pulse period.

14Fig. 18. instantaneous rate of CO production at half2

hour intervals, calculated from data plotted in

Fig. 17.

14 1419. Evolution of CO from guanidino- C-arginine by2

Fj g»

sugarcane cells. The experiment was conducted ex-

actly as the continuous-feeding, experiment de-

scribed in Chapter IV except that the isotope in

14this case was guanidino- C-arginine rather than

14 14ureido- C-citrulline. A shows CO production;

2

8 shows accumulation of label in cells

Fig. 17. Decarbamoylation of ureido- C-citrulline in a14

pulse-label experiment. Graph shows the cumula-

80

CO + NH

amoylphospha te

nthe.tase

2ADP + P.i

carbamoylphosphate

urea

ornithi

hine

carbamoylasear gina

arginine aspartate, ATP

osucci nate

etase

PP.

fumarate

argini

lyase arginin

The Ornithine Cycle in Animals

Figure 1

81

<DaJbO W

p b04JK

D

0 colcd«J

8 t0cd

bO

6 c3tdI

0

8

ed

cd

bQ

0 5A t

C3

04 CL S

0

I I

I I I I I I

I I I 1 I I I I I I 1I I I I II I I I I II I I t

QJ K

~ I

axginine

e desiminase

NH

citrulline

e transcarbameylase

carbamoylphq sphate

arnithine ADP

bamate kinase

ATP

CO , NH

The Arginine Desiminase Pathway

Figure 3

arginine

CO

agmatine

NH

N-carbamoylputrescine

CO, NH putrescine

polyamines

The Agmatine Pathway in Higher Plants

Figure 4

84

acetyl-CoA

Glutamate N-acetylglutamate

ATP

ADP

N-acetyl-0-glutamylphosphate

NADPH

NADP

N-acetyl-0-glutamylsemialdehyde

glutamate

g-ketoglutarate

N � acetylornithine

glutamate

N-acetylglutama

Ornithine

Ornithine Biosynthesis in Chlam 'domonas

Figure 5

85

O 0

OO 0 O O

OOOO O Ol

~~99t QO! 0<0

CS

aa l-

CO

IZ

86

O O

O O 040 0 0 0

~~ 99tr'0 0! OLO

U

LaJ

'ClO ~

C!

.C:

elCL

O OD O

«eat 0'0! QJ.O

~ OAI>A

7.0 8.0

PH

8.5

90

l00

~ BO

E60

E 40

20

i-

O M 6.0 65 70 75 8.0pH

IOO

~ ~ CJ

E

50

O.

CPI-

O 04 6'

Qrnithine mM!

92

i~[83 tmM Ornithine!

93

IO

FIg. IBb

5

ll'[S] mM Ornithine!

: 96

I

IA O !CQ

E CL

!

I/ [Sj mM Citrulline!

97

.4

I/ S rnM Citrulline!

Fig. 15b

2.I

a tA

o~

O OCC p7'

0

Time hours!

I

Time hours !

Fig. l8

8

3

Time hovrs!

LITEPATURK CITED

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1 o r . 1 9 6:-9,.:.":;,:.;:P a.r t i a 1

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