Plant Physiol. (1973) 51, 863-867

Isocitrate Lyase in Green Leaves1Received for publication September 19, 1972

H. R. GODAVARI, S. S. BADOUR, AND E. R. WAYGOODDepartment of Botany, University of Manitoba, Winnipeg, Manitoba. R3T 2N2, Canada

ABSTRACT

Isocitrate lyase (EC 4.1.3.1) has been demonstrated incrude dialyzed extracts of healthy spinach (Spinacia oleracea)leaves from commercial sources and wheat (Triticum aestivum)and maize (Zea mays) leaves stored in darkness in the coldroom for 1 week. The products of the reaction were identifiedas glyoxylate and succinate, the former by its phenylhydrazone,and the latter traced by isotopic labeling and cochromatogra-phy. Fresh spinach extracts contain a mixture of at least twoendogenous inhibitors of isocitrate lyase activity and one ofthem is proteinaceous. The endogenous inhibitor(s) is ther-mostable and retains 50% of its inhibitory effect even afterboiling for 10 minutes. Dark starvation of the leaves removesthe inhibition, due possibly to autolysis of the inhibitor(s).The inhibitor(s) can also be removed by filtration throughSephadex gels. The crude extract from spinach shows doublepH optima in phosphate buffer at pH 7.4 and pH 8.0. Theapparent Km at pH 7.4 was 0.1 mM. Oxaloacetate, DL-malate,succinate, 3-phosphoglycerate, and glycolate at 10 mM con-centration inhibited, but ribulose 1, 5-diphosphate activatedenzymic activity.

Isocitrate lyase (EC 4. 1 .3. 1) catalyzes the reaction

isocitrate < glyoxylate + succinate

and has been reported to be widely distributed (26). Isocitratehas been demonstrated in crude extracts of various unicellularalgae when forced to grow on acetate as the sole carbon source(10, 11). However, substantial activity has been observed inphotoautotrophically grown algae with CO2 as the sole carbonsource (2, 3, 7, 12, 27). Harrop and Kornberg (12) suggestedthat the presence of isocitrate lyase in autotrophic green algaewould not necessarily implicate the operation of glyoxylatecycle to replenish the C4 acids as in algae or bacteria grown onacetate (14), since these acids could arise by the /8-carboxyla-tion of phosphoenolpyruvate. This was supported further bythe reports of Foo et al. (7) and Badour and Waygood (2).

Isocitrate lyase is present in glyoxysomes of fatty seedlings(8, 13, 15, 16, 19) but is considered to be lost during growthand maturation (4, 5, 24). Isocitrate lyase activity has notbeen detected in green leaves (5), and the assumption of itsabsence seems plausible, since there has been no metabolicindication of its presence in green leaves (17, 18).The enzyme could be induced continuously in autotrophic

'This work was supported by Grants-in-Aid (A2698 and A6720)from the National Research Council of Canada.

Chiorella solely by placing the cells in the dark (3). Recently,Foo et al. (7) have shown that glycolate, 3-phosphoglycerate,and oxaloacetate are powerful inhibitors of isocitrate lyasefrom photoautotrophically grown Chlamydomonas segnis(Gloeomonas sp.). Inhibition of photosynthesis in C. segnis byDCMU (1) led to a 2-fold increase in the level of isocitratelyase as compared with photosynthesizing cells, whereas inPHMS -treated cells where glycolate accumulates, the enzymelevel is substantially decreased.

These results suggested a re-examination of the enzyme ingreen leaves, since in actively photosynthesizing green leavesisocitrate, lyase may be present but may be undetected in crudeextracts with a relatively high concentration of photosyntheticmetabolites.

MATERIALS AND METHODS

All plants were grown in the greenhouse with the exceptionof spinach (Spinacea oleracea) which was obtained from thelocal market. Healthy, turgid green leaves with no visible infec-tion or disease were used throughout. Commercial spinachleaves were surface sterilized by repeated washing in steriledistilled water and homogenized in a Waring Blendor understerile conditions. The homogenate was inoculated on agarplates to detect any internal agents of infection and only a fewbacterial colonies appeared, indicating that our plant materialwas virtually free of bacterial or fungal infection.Enzyme Extraction and Gel Filtration. The leaves were ex-

tracted by grinding in mortar and pestle with 0.1 M KH2PO,buffer, pH 7.3 (w/v 1:1). Approximately 0.5 g of acid-washedsand was added to the mortar to facilitate grinding. The fil-trate, after passing through four layers of cheesecloth, wascentrifuged at 35,000g for 30 min, and the clear supernatantwas used for assay or for further purification. Alternately,bulk quantities of commercial spinach leaves were passedthrough a vegetable juice extractor and an equal volume of0.1 M KH2PO, buffer at pH 7.3 was added to the juice. Thisextract was then centrifuged at 35,000g for 30 min, and theclear supernatant was used for assay or for further purification.An aliquot of the clear supernatant (crude homogenate) ob-

tained in the previous step was routinely dialyzed overnight(12-16 hr) against 40 vol of TEM buffer and assayed.The supernatant was fractionated with solid (NH4)-S04 be-

tween the limits of 40 to 60% saturation, and the protein wassuspended in a minimum volume of TEM buffer, pH 7.3, con-taining 10 mm dithiothreitol. The protein solution was filteredthrough a column of either Sephadex G-15 or G-100 (5 X 10cm) pre-equilibriated with TEM buffer. The effluent was col-lected in 5.0-ml aliquots, and the protein emerged in two

'Abbreviations: DNP: 2,4-dinitrophenyl hydrazone; PHMS:2-pyridylhydroxymethanesulfonic acid; TEM: 14 mm tris + 1.0 mMEDTA + 1.0 mM mercaptoethanol.

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Table I. Isocitrate Lyase Activity in Preparationzs ofVarious Plantt Species

All plants were grown in the greenhouse under natural daylightconditions except for spinach obtained from commercial sources.Only laminae were used to prepare the enzymes, as described inthe text. Assay system is as described in text using 10 ,.moles ofDL-isocitrate.

Approimate Crude Afe GlPlant Species Approximate Homo- Dialyfis FiltrateAe genateDilssFtre

utzoles glyoxylate,/nzgprotein.*lM

Bryopihyllum daigremontea- 3-4 months I- - 2.6niuim (Ham. Perr.) Berger

Linuirnn usitatissimnnni L. cv. 4 weeks - - 2.3Bison

Nicotiania rabacum L. 6 weeks - 9.0Spiniacia oleracea L. (com- 11.6 34

mercial)Triticumn aestivuin L. cv. 3 weeks - - 3.6Selkirk

Zea mays L., cv. Earliking 4-6 weeks - - 9.6

1 Not detectable.

major peaks with isocitrate lyase activity appearing in thesecond peak.

Assay. The enzyme was assayed according to the method ofRao and McFadden (25) which is based on the formation of ared colored glyoxylate-phenylhydrazone-ferricyanide complexwith Ynax 520 nm. The assay system contained in ,umoles: MESbuffer (pH 7.3) 50; MgCl2, 7.5: GSH, 2.0; DL-isocitrate, 2 to10 with 0.5 ml of enzyme in a final volume of 1.3 ml. Thereaction was initiated by the substrate after a preincubationof the enzyme and cofactors for 10 min at 30 C. The completeassay system was routinely incubated in duplicate, and the re-action was terminated by adding 0.4 ml of 1.0 M oxalic acid.After addition of 0.1 ml 5% (w/v) phenylhydrazine-HCl, themixture was brought to boiling within 1 min, quickly cooled inice, and 1.8 ml of concentrated HCl added. A red color de-velops if any glyoxylate is formed on adding 0.1 ml of 25%(w/v) K3Fe(CN)6. In studies dealing with the effects of metabo-lites on enzyme reaction, test compounds were preincubatedwith enzyme and cofactors for 10 min before initiating thereaction.

Protein. Protein was determined according to Lowry et al.(20).Thin Layer Chromatography. Glass plates (20 X 20 cm)

coated with MN 300 cellulose, 250 ,. thick, were used forchromatographic analysis of the enzymic reaction products af-ter deproteinization of the reaction mixture. For separation ofDNP derivatives, the solvent system t-amyl alcohol-ethanol-H20 (5:1:4) was employed. The characteristic colors of theDNP derivatives on the chromatogram were visualized byethanolic 4 N NaOH spray. Organic acids were separated inpropyl acetate-formic acid-H20 (11:5:3), and the spots werelocated either after aniline-xylose spray and heating at 110 Cor on x-ray film if labeled DL-isocitrate was the substrate.Column Chromatography. Labeled succinate and isocitrate,

in the deproteinized reaction mixture, were separated onDowex AG-1 X 10 acetate columns (1 X 9 cm) prepared ac-cording to Zelitch (29).

RESULTSDetection of the Enzyme. In the initial experiments with the

crude homogenate, no activity was observed in all leaves tested

Plant Physiol. Vol. 51, 1973

with the exception of spinach which gave the colored complexin both the complete system and the control without isocitrate.After dialysis for 4 hr against TEM buffer (pH 7.3) onlyspinach extracts showed activity, as indicated by the formationof the red colored complex, and only in the complete reactionmixture. Even following gel filtration, the formation of thereddish colored complex always occurred in the control withoutisocitrate, but to a lesser extent than in the complete system.Thus the calculated specific activities for the enzyme after gelfiltration of leaf extracts of the plants indicated in Table Iwere very low (2.3-9.6 nmoles/min-mg protein) as comparedwith that of dialyzed crude extract of spinach leaves (12nmoles/min-mg protein). However, even this high value ob-tained with the spinach leaf enzyme is about the same as thelowest values (for soybean cotyledons) reported by Carpenterand Beevers (5). Nonetheless, detection of the enzyme wasconfirmed in all these plants by tracing its activity using 5, 6-'4CDL-isocitrate and identification of labeled succinate by radio-autography.

Demonstration of Isocitrate Lyase Activity in DarkenedLeaves. The presence of the enzyme in crude extracts of spin-ach which presumably had been stored in darkness and coldin the supermarket suggested that it was these prior environ-mental conditions that had resulted in their higher activityeither by removal of an inhibitor or by induction.We have exposed detached wheat and maize leaves to dark-

ness within plastic bags in the cold room for 1 week, and theextracts of these leaves behave in a similar manner to spinachextracts. In undialyzed extracts of maize and wheat, the reddishcolored complex was always formed in both the control andcomplete systems, but after dialysis it appears only in the com-plete system indicating enzymic activity. Progress curves of thereaction using dialyzed leaf extracts are shown in Figure 1with the formation of approximately 0.9 ,tmole of glyoxylatewhich represents a stoichiometric utilization of the threo-Ds-iso-citrate in 2.0 ,umoles of the DL-isocitrate added. Table II showsthat Mg2+ and GSH are essential for maximum activity.

Identification of the Product. The products of the reactionwere determined by TLC of the 2,4-dinitrophenylhydrazonederivative of glyoxylate (Fig. 2) which produced a brick-redcolor with 2 M NaOH and showed an absorption spectrum witha maximum at 450 nm identical with that of authentic gly-

1.6

1.4

1 2

E

.4-

1.0

0.8

0.6

0.4

0.2

0

0.9 F

x0

0

0.6

E

0.3

0 1 0 20 3 0 40 50 60 70 80 90

T I ME (min)FIG. 1. Time course for the isocitrate lyase activity. Assay sys-

tem is as described in the text using 2 uemoles of DL-isocitrate. Thereaction was initiated by the addition of substrate after preincuba-tion for 10 min. Crude homogenates dialyzed overnight against 40volumes of TEM buffer. 0: Spinach enzyme containing 3.6 mg ofprotein. 0: wheat leaf enzyme containing 2.2 mg of protein.

GODAVARI, BADOUR, AND WAYGOOD

I

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ISOCITRATE LYASE IN GREEN LEAVES

Table II. Effect of Cofactors otn Isocitrate Lyase Activityin Spinach anid Wheat Leaf Extracts

Assay system is as described in text using 10 1umoles of DL-isocitrate. Preincubation was 10 min, and incubation was 30 min.Crude homogenates were dialyzed overnight against 40 volumesof TEM buffer.

Omissions Spinach WVheat

% of control

Control 100' 1002Mg2+ 49 83GSH 77 85Mg'+ and GSH 31 78Enzyme (boiled) 0 0

1 11.6 nmoles glyoxylate/mg protein-min.2 12 nmoles glyoxylate/mg protein-min.

oxylate. In addition, enzymically produced glyoxylate was re-duced by NADH to glycolate (identified chromatographically)using the same dialyzed crude leaf extract. The other reactionproduct, succinate, was identified by radioautography as de-scribed before (Fig. 3).Under the conditions of assay, isocitrate was apparently

glyoxylate. Assay system is as described in text using 10 umoles1 2 3 4 5 6 7 8 of DL-isocitrate. 1: Glyoxylate DNP (marker); 2: cx-ketoglutarate-

DNP (marker); 3: spinach leaf enzyme without isocitrate; 4, 5:FIG. 2. TLC of the 2,4 dinitrophenyl hydrazone derivatives of spinach leaf enzyme with isocitrate; 6: wheat leaf enzyme without

authentic glyoxylate, a-ketoglutarate, and enzymically produced isocitrate; 7, 8: wheat leaf enzyme with isocitrate.

S 5 10 15 30 60 90 M IB KB

T I M E (min)FIG. 3. Thin layer autoradiogram of the '4C-succinate (--) formed in a time course for isocitrate lyase activity. Assay system is as described in

text using 10 Ajmoles of DL-isocitrate-5, 6-14C and spinach leaf enzyme. Crude homogenate dialyzed overnight against 40 volumes ofTEM buffer.

I: DL-Isocitrate-5,6-_4C; K: ai-ketoglutarate (U)-_4C; S: succinate-2,3-'4C; M: mixture of markers with boiled enzyme; IB: DL-isocitrate-5,6-_4Cwith boiled enzyme; KB: a-ketoglutarate (U)-14C with boiled enzyme.

*

^ * ', * T

I I I I I I

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865Plant Physiol. Vol. 51, 1973

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GODAVARI,BADOUR, AND WAYGOOD >I .iA' ' Plant Physiol. Vol. 51 1973

Table III. Stoichiometry of the Isocitrate Lyase Reaction

Assay system is as described in text. Ten,umoles of DL-isocitratewere added to the reaction mixture including 1.3 mg/protein.Commercial spinach crude homogenate dialyzed overnight against40 volumes of TEM buffer was used. Glyoxylate was determinedcolorimetrically (at 520 nm); '4C-succinate was separated onDowex AG-1 X 10 acetate columns by 4 N acetic acid.

ProductsTime of Incubation

Glyoxylate Succinate

min jvnzoles fornmed15306090

0.350.931.74

0.450.661.202.43

utilized by the lyase rather than isocitrate dehydrogenase, sinceno traces of its product, a-ketoglutarate, could be detected.The DNP-derivative of a-ketoglutarate migrated ahead of gly-oxylate-DNP on thin layer chromatograms and produced anolive green color with 4 M NaOH (Fig. 2). Obviously, a-keto-glutarate was not one of the products (Fig. 2). This was furtherconfirmed by the use of 5,6-'4C DL-isocitrate. As shown inFigure 3, succinate was the only product labeled with notrace of a-ketoglutarate.

Stoichiometry. Stoichiometry of the reaction was determinedusing spinach extracts incubated with 10 ,.umoles of DL-iSocit-rate, and the products were determined on parallel tubes.Glyoxylate was measured according to the procedure of Raoand McFadden (25) as described in "Materials and Methods."DL-Isocitrate-5, 6-'4C (5.9 ,uc/mmole) was incubated with theenzyme in parallel, and the products were separated quanti-tatively on Dowex AG 1 X 10 acetate columns prepared ac-

0.6

0.3

Ec 0.2

0.1

0 0.2 0.4 0.6 0.8

DAY-OLD SPINACH EXTRACT (ml.)

FIG. 4. Inhibition of isocitrate lyase (spinach) by dialyzed crudeextracts of fresh spinach. Assay system is as described in text using10 ,umoles of DL-isocitrate. Crude homogenates dialyzed overnightagainst 20 volumes of TEM buffer.

Table IV. Inzhibition of Isocitrate Lyase Activity by antEndogenous Inhibitor from Fresh Spinach Extract

Assay system is as described in text using 10 ,moles of DL-

isocitrate. Preincubation was 10 min, and incubation was 30 min.The spinach enzyme was dialyzed. Five ml of dialyzed extract

of fresh spinach were brought to 90%7 saturation with cold ethanol.

It was stored in the cold for 3 hr and centrifuged at 48000g for

10 min. The precipitate was redissolved in 5 ml of 0.1 M MES

buffer at pH 7.3. This was the alcohol-insoluble fraction. The

supernatant was dried down, and the residue was dissolved in 5

ml of 0.1 M MES buffer at pH 7.3, and this was the alcohol soluble

fraction. The spinach enzyme was a crude homogenate dialyzedovernight against 20 volumes of TEM buffer.

Additions AA at 520 nm In}

NoneFresh extractAlcohol-insoluble fraction

Alcohol-soluble fractionBoiled extract (10 min)

0.4150.0560.2020.2850.260

0.1

0.5 F

0.4

Ec 0.3

0.2

0.3

X

0.2 0

-1

0

EO..

0.1

6 7 8 9

p H

FIG. 5. Effect of pH on isocitrate lyase from spinach determinedin phosphate buffer. Assay system is as described in text using 10,umoles of DL-isocitrate. Crude homogenate dialyzed overnightagainst 40 volumes of TEM buffer.

Table V. Inihibitionz of Isocitrate Lyase Activity bySonme Metabolites

Assay system is as described in text using 10 ,moles of DL-

isocitrate. Preincubation was 10 min. The spinach enzyme was

crude homogenate dialyzed overnight against 40 volumes ofTEM buffer.

MIetabolite - Substrate + Substrate Inhibition

(10 mm)

hibition NoneGlycolate

Co0 DL-Malate0 Oxaloacetate87 3-Phosphoglycerate52 Succinate31 None43 Ribulose-1,5 diP

A

0.2000.8000.2551.800.3500.1750.4300.336

0.7601.0390.2931.9650.5140.3430.8521.152

0

6093707070

2-fold activation

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ISOCITRATE LYASE IN GREEN^LEAVES

cording to Zelitch (29). Radioactivities of the succinate formed,and the residual isocitrate was determined by liquid scintilla-tion. There is a parallel increase in the amounts of glyoxylateand succinate formed with time (Table III). The observed ratioof 1.7 /cmoles of glyoxylate to 1.2 ,umoles of succinate formedafter 60 min is in good agreement with the theoretical valueof 1: 1 considering the crude nature of the enzyme preparation.Endogenous Inhibitor(s) of Isocitrate Lyase. Isocitrate lyase

activity could be detected in crude dialyzed extracts of com-mercial spinach, but not in extracts of freshly harvested spin-ach. As shown in Figure 4, crude extracts prepared from freshspinach inhibited the lyase activity of the dialyzed extracts ofcommercial spinach. This indicates that an inhibitor, originallypresent in the green leaves or formed during homogenization,masks the enzyme activity. The inhibitory effect appears to beattributable to at least two inhibitors (Table IV); one is alcohol-insoluble, presumably a protein, and the other is alcohol-solu-ble. The sum total of inhibition caused by the two fractions(Table IV) nearly equals the inhibition caused by the freshextract, indicating that both fractions possibly exerted an addi-tive inhibitory effect on isocitrate lyase activity and appear tobe partially thermostable retaining 50% of the inhibitory effecteven after boiling for 10 min.Enzyme Properties. Dialyzed extracts of commercial spinach

were used to study some properties of this enzyme. As shownin Figure 5, the dialyzed crude preparation in phosphate bufferat 30 C showed two pH optima; one at pH 7.4 and the otherat pH 8.0. This may suggest the presence of two isozymes inthe leaf extract. The enzyme has shown an apparent Km of 0.1mm at pH 7.4 in phosphate buffer. The enzyme is susceptible toend product inhibition by succinate (Table V) as well as inhibi-tion by oxaloacetate, DL-malate, glycolate, and 3-phospho-glycerate at 10 mm in a manner similar to that reported foralgal enzyme (7). It also showed an unusual activation byribulose-1, 5-diP.

DISCUSSIONThe present work reported previously (9) has provided evi-

dence for the presence of (a) isocitrate lyase in green leavesand (b) the presence of endogenous inhibitor(s) of isocitratelyase. The enzyme in intact green leaves may be operating asin green algae to provide succinate for succinyl-CoA forma-tion, if the latter were not produced via a-ketoglutarate de-hydrogenase, as suggested by Pearce et al. (21, 22) and Fooet al. (7). Alternately, the glyoxylate may undergo condensa-tion and decarboxylation with a-ketoglutarate via glyoxylate-a-ketoglutarate carboligase shown to be present in higher plants(6). Recently Zelitch (30) reported the synthesis of glycolatefrom acetate-2-14C and pyruvate-3-'4C in maize and tobaccoleaves. This suggests a possible role for isocitrate lyase in theformation of glyoxylate which may then be reduced to gly-colate. Accumulation of glyoxylate in excised wheat leavesduring anaerobic starvation in darkness reported by Krupkaand Towers (17, 18) should be reconsidered in this light.

Evidently, the presence of an endogenous inhibitor(s) of iso-citrate lyase in fresh green spinach prevents the detection ofthe enzyme in extracts of green leaves. Moreover, the enzymeis also susceptible to inhibition by various metabolites. If theinhibitory effect is partially attributable to photosynthates, thepool size of the latter would decrease by prolonged storageunder darkness. Dark starvation may also result in proteinautolysis as concluded by Postius and Jacobi (23). The darkstarvation would decrease or even degrade the inhibitor(s) orthe enzymes mediating the synthesis of these inhibitors.

In the present work, it has also been shown that the in-hibitor complex can be removed either by gel filtration or by

autolysis under dark starvation, possibly it is reversible. Theease of removal indicates that it is a rather loose complex ofenzyme and inhibitor. It is possible that the enzyme-inhibitorcomplex in vivo is readily dissociable thus giving the plant aflexible directional control of the flow of isocitrate, as signalledby various allosteric activators and inhibitors of the enzyme.

LITERATURE CITED

1. BADOUR, S. S., S. K. Foo, AND E. R. WAYGOOD. 1972. Levels of isocitrate lyaseduring the cell cycle of Chlamydomonas segnis in synchronous cultures.Proc. Can. Soc. Plant Physiol. 12: 10-11.

2. BADOUR, S. S. AN-D E. R. WAYGOOD. 1971. Glyoxylate carboxy-lyase activityin the unicellular green alga Gloeomonas sp. Biochim. Biophys. Acta 242:493-499.

3. BAECHTEL, F. S., H. A. HopKiN-s, AN-D R. R. SCHMIDT. 1970. Continuousinducibility of isocitrate lyase during the cell cycle of the eucaryoteChlorella. Biochim. Biophys. Acta 217: 216-219.

4. BEEVERS, H. 1969. Glyoxysomes of castor bean endosperm and their rela-tion to gluconeogenesis. Ann. N.Y. Acad. Sci. 168: 313-324.

5. CARPENTER, W. D. AND H. BEEVERS. 1959. Distribution and properties ofisocitritase in plants. Plant Physiol. 34: 403-409.

6. DAVIES, D. D. AND P. KENWORTHY. 1970. a-Oxoglutarate: glyoxylate car-boligase activity of plant mitochondria. J. Exp. Bot. 21: 247-257.

7. Foo, S. K., S. S. BADOUR, AN-D E. R. WAYGOOD. 1971. Regulation of isocitratelyase from autotrophic and photoheterotrophic cultures of Gloeomonas sp.Can. J. Bot. 49: 1647-1653.

8. GERHARDT, B. P. AND H. BEEVERS. 1970. Developmental studies on gly-oxysomes in Ricinus endosperm. J. Cell Biol. 44: 94-102.

9. GODAVARI, H. R., S. S. BADOUR, AND E. R. WAYGOOD. 1972. Isocitrate lyasein green leaves. Proc. Can. Soc. Plant Physiol. 12: 26-27.

10. GOILDING, K. H. AND MI. J. MERETT. 1966. The photometabolism of acetateby Chlorella pyrenoidosa. J. Exp. Bot. 17: 678-689.

11. HAIGH, W. G. AND H. BEEVERS. 1964. The occurrence and assay of isocitratelyase in algae. Arch. Biochem. Biophys. 107: 147-151.

12. HARROP, L. C. AND H. L. KORNBERG. 1966. The role of isocitrate lyase in themetabolism of algae. Proc. Roy. Soc. B 166: 11-29.

13. KAGAWA, T. AN-D H. BEEVERS. 1970. Glyoxysomes and peroxisomes in water-melon seedlings. Plant Physiol. 46: S-38.

14. KOR'BERG, H. L. 1965. Regulations chez les micro-organismes. CentreNationale de la Recherche Scientifique Colloques Internationaux. Paris. 124:193-207.

15. KORNBERG, H. L. and H. BEEVERS. 1957. A mechanism of conversion of fatto carbohydrate in castor bean. Nature 180: 35-36.

16. KORNBERG. H. L. A.ND H. BEEVERS. 1957. The glyoxylate cycle as a stagein the conversion of fat to carbohydrate in castor bean. Biochim. Biophys.Acta 26: 531-537.

17. KRUPKA, R. M. AND G. H. N. TOWERS. 1958. Studies of the keto acids ofwheat. I. Behaviour during growth. Can. J. Bot. 36: 165-177.

18. KRI-PKA, R. M. AND G. H. N. TOWERS. 1958. Studies of the keto acids of wheat.II. Glyoxylic acid and its relation to allantoin. Can. J. Bot. 36: 179-186.

19. LON-GO, G. P. AND C. P. LONGO. 1970. The development of glyoxysomes in maizescutellum. Plant Physiol. 46: 599-604.

20. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951.Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

21. PEARCE, J. AND N. G. CARR. 1967. An incomplete tricarboxylic acid cycle inthe blue-green alga Anabaena variabilis. Biochem. J. 105: 45.

22. PEARCE, J., C. K. LEACH, AND N. G. CARR. 1969. The incomplete tricarboxylicacid cycle in the blue-green Anabaena variabilis. J. Gen. Microbiol. 55:371-378.

23. POSTIUS, S. AND G. JACOBI. 1971. Dark starvation and chloroplast function.I. The decrease of enzyme activities correlated with NADP reduction andtheir regeneration by light. Planta 99: 222-229.

24. PRICE, C. A. 1970. Molecular approaches to plant physiology. McGraw-Hill,Inc., New York. pp. 32-33.

25. RAO, G. R. AND B. A. McFADDEN. 1965. Isocitrate lyase from Pseudomonasindigofera. IV. Specificity and inhibition. Arch. Biochem. Biophys. 112: 294-303.

26. TOLBERT, N. E. 1971. Microbodies-peroxisomes and glyoxysomes. Annu. Rev.Plant Physiol. 22: 45-74.

27. WIESSN-ER, W. 1968. Enzymaktivitiit und Kohlenstoffassimilation beiGrunalgen unterschiedlichen ernahrungsphysiologischen Typs. Planta 79:92-98.

28. WIESSNER, W. AND A. KUHL. 1962. Die Bedeutung des Glyoxylsaure-Zyklusfur die Photo-assimilation von Acetate bei Phototrophen Algen: im Vor-triage aus dem Gesamtgeibiet der Botanik, N.F., Nr. 1, S. 102-108. Stuttgart:Gustav Fischer.

29. ZELITCH, I. 1958. The role of glycolic acid oxidase in the respiration of leaves.J. Biol. Chem. 233: 1299-1303.

30. ZELITCH, I. 1972. Alternate pathways of glycolate synthesis and their relationto photorespiration in tobacco and maize leaves. Plant Physiol. 49: S-329.

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