Oxidation by Spinach Leaf Mitochondria' Received for Publication May 28, 1980 and in Revised Form October 1, 1980

Plant Physiol. (1981) 67, 467-469 0032-0889/81/67/0467/03/$00.50/0

Role of Glutamate-oxaloacetate Transaiinase and Malate Dehydrogenase in the Regeneration of NAD+ for Glycine Oxidation by Spinach leaf Mitochondria' Received for publication May 28, 1980 and in revised form October 1, 1980

ETIENNE-PASCAL JOURNET, MIcHEL NEUBURGER, AND ROLAND DOUCE Physiologie Cellulaire Vigeitale, De'partement de Recherche Fondamentale/Biologie Vigitale, Centre d'Etudes Nucliaires et Universiti Scientfifque et Midicale de Grenoble, 85X 38041 Grenoble Cedex, France

ABSTRACT v) defatted BSA (medium A). The 02 concentration in air-satu- rated medium A was taken as 240 pM (7). During glycine oxidation by spinach leaf mitochondria, oxygen con- Assay of Metabolic Products. Metabolic products were rou- sumption showed a strong and transient inhibition upon addition of tinely assayed in a system containing medium A, 5 mm glycine, oxaloacetate or aspartate plus a-ketoglutarate. During the course of the and known amounts of mitochondrial protein. The reaction was inhibition, aspartate and a-ketoglutarate were stoichiometricafly trans- initiated by the addition of aspartate and a-ketoglutarate. Incu- formed into malate and glutamate. bation temperature was 25 C. At various times, 1-ml aliquots were It is concluded that oxaloacetate formed by transamination is reduced taken and added to 0.3 ml ice-cold 20% (v/v) HC104. The samples by the malate dehydrogenase, which aglows the regeneration of NAD' for were quickly neutralized with 5 N KOH and centrifuged for 5 min glycine oxidation and, thus, by-passes the respiratory chain. Efficiency of at 5,000g to remove KC104. The supernatant was used for L- a malateglutamate/aspartate-a-ketoglutarate shuttle upon illumination malate, glutamate, a-ketoglutarate, and serine determination. Si- and under in vivo conditions is discussed. multaneously the 02 consumption of 1-ml aliquot was measured. L-Malate was assayed by the method of Gutmann et al. (8) and a-ketoglutarate by the procedure of Bergmeyer et al. (2). Gluta- mate and serine were analyzed in sodium citrate buffers with an amino-acid analyzer (model 118 C, Beckman Instruments, Palo Alto, CA). It is established that isolated spinach leaf mitochondria are Niltochondrial Protein Determination. Total protein was deter- capable of oxidizing glycine, an important metabolite of photo- mined by the Folin-Ciocalteu phenol reagent (15). If we assume respiration (18, 21), and that this oxidation which is very rapid (5) a protein to Chl ratio of 7 in broken thylakoids (14), the amount is coupled to the synthesis of 3 molecules ATP (5). During the of mitochondrial protein has to be corrected for the contribution course of glycine oxidation, 2 mol glycine are converted to 1 mol of the broken thylakoids. ChM was extracted from the mitochon- each of serine, NH3, and CO2 in a reaction catalyzed by glycine drial pellet in 80% acetone and measured according to Arnon (1). synthase and hydroxymethyltransferase (18). The object here is to demonstrate the role of a malate dehydro- RESULTS genase (L-malate:NAD' oxidoreductase, EC 1.1.1.37) and a glutamate-oxaloacetate transaminase (L-aspartate:a-ketoglutarate Figure 1 indicates that spinach leafmitochondria oxidize glycine aminotransferase, EC 2.6.1.1.) in the regeneration of NAD+ for as substrate very rapidly with a good respiratory control. glycine decarboxylation. ADP:O ratios observed are close to 3 (see also ref. 5). Figure 2 shows the effect of oxaloacetate on the respiratory rates with MATERIALS AND METHODS glycine as substrate in spinach-leaf mitochondria. When oxaloacetate is added to state 3, a clear inhibition of the respiration rate Preparation of Mitochondria. Young spinach (Spinacia oleracea occurs whicn is gradually reversed. Enzymic analysis showed that, L.) leaves (0.8 kg after deribbing) were cut into 3 liters chilled during the time of inhibition, the oxaloacetate is converted to medium containing 0.3 M mannitol, 4 mm cysteine, 1 mm EDTA, malate (results not shown), a conversion that is dependent on the 20 mM pyrophosphate buffer (pH 7.5), 0.2% (w/v) defatted BSA, intramitochondrial NADH generated during the course ofglycine and 0.6% (w/v) insoluble PVP. The leaves were disrupted at low oxidation. These results are in good agreement with those of speed for 2 s in a 1-gallon Waring Blendor, and mitochondria Douce and Bonner (3) and Woo and Osmond (20). were isolated as fast as possible by differential centrifugation It is also possible that a-ketoglutarate undergoes transamination according to the method of Douce et aL (4, 5). with aspartate leading to the formation of glutamate and oxaloac- 02 Uptake Measurements. Mitochondrial respiration was mea- etate inasmuch as the enzyme involved (glutamate-oxaloacetate sured polarographically using a Clark 02 electrode in a closed cell transaminase, EC 2.6.1.1.) is present in the mitochondrial matrix at 25 C. 02 consumption measurements were performed in a space (for a review, see ref. 17). Consequently, oxaloacetate thus reaction medium (1 ml) containing: 0.3 M mannitoL 5 mM MgC12, directly formed in the matrix space would oxidize intramitochon- 10 mm KCL 10 mm Na-phosphate buffer (pH 7.2), and 0.1% (w/ drial pyridine nucleotides, permitting the regeneration of NAD+ for glycine decarboxylation. Under these conditions, the respira- ' Supported in part by a research grant from the Centre National de la tory chain should be by-passed. Recherche Scientifique (ERA 847: Interactions Plastes-Cytoplasme-Mito- To verify this hypothesis, we followed the effect of aspartate chondries). and a-ketoglutarate on the respiratory rates with glycine as sub- 467 468 JOURNET, NEUBURGER, AND DOUCE Plant Physiol. Vol. 67, 1981

MW 10mM Glycee MW 15mM ADP MW lm Gycine MW 1.5MM ADP MW 1OmM Glycine lOmM Glycine 1OmM Glycine 140 pJM ADP \SmM Aspartale 5mM Aspartate 2mM 2mM 72 \a-Ketoglutarate a-Ketoglutarate

5 2mM 82mM 56 5mMAsPartate 80 5mM Aspartate 2\4 P/O. 2.5 \-Ketogkutarate a-Ketoglutarate 85\ 20 MO280 2

,min 72 86 pH pH 7.2 249 FIG. 3. Effect of a-ketoglutarate and aspartate on glycine oxidation by 82\ mitochondria isolated from spinach leaves. Numbers on traces refer to nmol 02 consumed/min -mg protein. MW, washed mitochondria. 21 20 jM 02 7 I MW 5 mM Aspartate 1 min I 10 mM Glycine

51100 pM .-Ketoglutarate FIG. 1. Glycine oxidation by mitochondria isolated from spinach leaves. Numbers on trace refer to nmol 02 consumed/min mg protein. MW, washed mitochondria. 26

4 200 pM.-Ketoglutarate 15MM ADP MW 10mM Glycine 13\ I \ 300 pM .-Ketoglutarate - ~~~32\ 78100 Oxaloacetate 20pM02 pM -K 10 14 1min pH72 24 80 VM Oxaloacetate

FIG. 4. Effect of amounts on 14 limiting of a-ketoglutarate glycine oxidation by mitochondria isolated from spinach leaves. Numbers on traces 1 7 300pM Oxaloacetate refer to nmol 02 consumed/min -mg protein. MW, washed mitochondria. 20OpM 02 I 12 500 270 paM a-ketoglutamtr serine M1 1Xa 1mi 60 400

pH 7.2 / glutamate

FIG. 2. Effect oflimiting amounts ofoxaloacetate on glycine oxidation by mitochondria isolated from spinach leaves. Numbers on trace refer to nmol 02 consumed/min.mg protein. MW, washed mitochondria. 7E> 3100° strate in spinach leaf mitochondria. When added separately, aspartate and a-ketoglutarate have no effect on the rate of glycine oxidation (Fig. 3). In contrast, addition of 2 mM ae-ketoglutarate to spinach leaf mitochondria oxidizing glycine in the presence of 5 mm aspartate slows the rate of 02 consumption considerably and FIG. 5. Disappearance of added a-ketoglutarate (A), production of vice versa (Fig. 3). However, during the inhibition of 02 con- malate Q), glutamate (Is) and serine (*), and 02 consumption (0) during sumption induced by aspartate and a-ketoglutarate, the transfor- glycine oxidation by mitochondria isolated from spinach leaves. The mation of glycine into seine keeps going (see Fig. 5). standard assay solution was used with 1.17 mg mitochondrial protein/ml, After addition of a small amount of a-ketoglutarate to mito- 10 mm glycine, and 5 mm aspartate. Final pH was 7.2. The fmal volume chondria-oxidizing glycine in the presence of 5 mM aspartate, a of the reaction medium was 15 ml. clear inhibition of the respiration rate occurs, which is gradually reversed (Fig. 4). The addition of twice the initial amount of a- ketoglutarate and aspartate are converted to 1 mol each of malate ketoglutarate again causes an inhibition of the oxidation rate, and and glutamate (Fig. 5). Under these conditions, neither malate nor the time period during which 02 consumption is stopped is twice glutamate are further metabolized (results not shown). The same the first one (Fig. 4). Enzymic analysis showed that, during the mechanism of a-ketoglutarate/aspartate inhibition pertains to the time of inhibition, a-ketoglutarate disappears gradually and is oxidation of malate (at pH 6.5) and malate-pyruvate (results not converted, as expected, to malate and glutamate. One mol of a- shown). Plant Physiol. Vol. 67, 1981 GLYCINE OXIDATION IN LEAF MITOCHONDRIA 469 tle (9, 12). The close association observed in vivo between mitochondria and peroxisomes could perhaps facilitate the direct transport of oxaloacetate between both cell organelles. Finally, these results raise the problem of aspartate transport into plant mitochondria. In contrast with the situation observed in animal mitochondria (13, 19), aspartate can enter plant mitochondria sufficiently rapidly to sustain a rapid transfer ofreducing equivalents out ofthe mitochondria. In fact, cytoplasmic aspartate concentration is high enough (16) to facilitate the rapid transfer of aspartate into mitochondria. Note Added in Proof. All these results have been confirmed 1 with intact mitochondria prepared from spinach leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. (Nishimura M., R. Douce, T. Akazawa. Plant Physiol, in press)

Acknowledgment-The authors are much indebted to Mrs L. Tranqui for the amino acid analysis.

LITERATURE CITED 1. ARNON DI 1949 Copper enzymes in isolated chloroplasts polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15 2. BERGMEYER HU, E BERNT 1974 2-Oxoglutarate. UV spectrophotometric determination. In HU Bergmeyer, ed, Methods of Enzymatic Analysis, Ed 2 Vol 3, Academic Press, New York, pp 1577-1580 3. DoUCE R, WD BONNER 1972 Oxaloacetate control of Krebs cycle oxidations in purified plant mitochondria. Biochem Biophys Res Commun 47: 619-624 4. DouCE R, EL CHRIsTENsEN, WD BONNER JR 1972 Preparation of intact plant mitochondria. Biochim Biophys Acta 275: 148-160 5. DouCE R, AL MooRE, M NEUBURGER 1977 Isolation and oxidative properties of FIG. 6. Scheme of photorespiratory pathway where the malate/aspar- intact mitochondria isolated from spinach leaves. Plant Physiol 60: 625-628 tate shuttle plays a predominant role in the exchange of reducing equiva- 6. DouCE R, M NEUBURGER 1979 R6le joue par les transporteurs d'anions des lents between mitochondria and peroxisomes. The reassimilation of NH3 mitochondries et des chloroplastes dans la photosynthese et la respiration. In C et Production Ed 2. formed during the course of glycine oxidation also is indicated (11). Costes, ed, Photosynthese Vegetale, Gauthier-Villars, Paris, pp 147-169 7. ESTABROOK RW 1967 Mitochondrial respiratory control and the polarographic DISCUSSION measurement of ADP/O ratios. Methods Enzymol 10: 41-47 8. GUTMANN I, AW WAHLEFELD 1974 L(-)-Malate. Determination with malate These results demonstrate that NADH produced during the dehydrogenase and NAD. In HU Bergmeyer, ed, Methods of Enzymatic Analysis, Ed 2 Vol 3. Academic Press, New York, pp 1585-1589 course oxidation can be reoxidized either the of glycine by respi- 9. HEBER U 1974 Metabolite exchange between chloroplasts and cytoplasm. Annu ratory chain or by oxaloacetate just formed by transamination. Rev Plant Physiol 25: 393-421 Both systems are competing at the level of the pyridine nucleotide 10. HEBER U, GH KRAUSE 1980 What is the physiological role of photorespiration? pool and that NAD+-linked glycine decarboxylase provides TIBS 5: 32-34 11. KEys AJ, IF BIRD, MJ CORNELIUS, PJ LEA, RM WALLSGROVE, BJ MIFLIN 1978 NADH for the reduction of oxaloacetate catalyzed by the mito- Photorespiratory nitrogen cycle. Nature 275: 741-743 chondrial malate dehydrogenase. It is probable that all the en- 12. KRAUSE GH, U HEBER 1976 Energetics of intact chloroplasts. In J Barber, ed, zymes involved in these reactions (glutamate-oxaloacetate trans- The Intact Chloroplast, Topics in Photosynthesis, Vol 1, Elsevier/North-Hol- aminase, malate dehydrogenase, glycine decarboxylase) exist as a land Biomedical Press, Amsterdam, pp 171-214 13. LA NouE KF, AC SCHOOLWERTH 1979 Metabolite transport in mitochondria. multienzyme complex, localized in the matrix space. Annu Rev Biochem 48: 871-922 As pointed out by Heber and Krause (10), intramitochondrial 14. LiLLEY RMcC, MP FITZGERALD, G RIENITs, DA WALKER 1975 Criteria of NADH can be reoxidized in vitro by the respiratory chain. How- intactness and the photosynthetic activity of spinach chloroplast preparations. ever, such a situation is improbable under in vivo conditions New Phytol 75: 1-10 15. LowRY because the ATP:ADP ratio is considerable upon OH, NJ ROSEBROUGH, AL FARu, RJ RANDALL 1951 Protein measure- cytoplasmic ment with the Folin-phenol reagent. J Biol Chem 193: 265-275 illumination (9). As a matter of fact, the rate of mitochondrial 16. MILLS WR, KW Joy 1980 A rapid method for isolation of purified, physiologi- electron transport is governed by the cytoplasmic phosphorylation cally active chloroplasts, used to study the intracellular distribution of amino potential (6). In addition, reducing equivalents formed inside the acids in pea leaves. Planta 148: 75-83 17. SCHNARRENBERGER H FOCK 1976 the course of must C, Interactions among organelles involved in mitochondria during glycine oxidation be photorespiration. In CR Stocking, U Heber, eds, Transport in Plants, Encyclo- transported to the peroxisomes for the reduction of hydroxypyr- pedia of Plant Physiology, New Series, Vol 3. Springer-Verlag, Berlin, pp 185- uvate (for a review, see ref. 17). The results presented here indicate 234 that this could be achieved by metabolic shuttles such as malate/ 18. TOLBERT NE 1971 Leaf peroxisomes and photorespiration. In MD HATCH, CB oxaloacetate (3) or malate/aspartate shuttles (Fig. 6). However, Osmond, RC Slatyer, eds, Photosynthesis and Photorespiration. Wiley-Inter- science, New York, pp 458-471 the transport of oxaloacetate between both cell organelles via the 19. WILLIAMSON JR 1976 Role of anion transport in the regulation of metabolism. cytoplasm is improbable inasmuch as oxaloacetate would be im- In RW Hanson, MA Mehlman, eds, Gluconeogenesis-Its Regulation in mediately converted into malate by the cytoplasmic malate de- Mammalian Species. Wiley-Interscience, New York, pp 165-220 hydrogenase because, under conditions of continuous photosyn- 20. Woo KC, CB OSMOND 1976 Glycine decarboxylation in mitochondria isolated from spinach leaves. Aust J Plant Physiol 3: 771-785 thesis, an excess of reducing equivalents is transferred to the 21. ZELITCH I 1971 Photosynthesis, Photorespiration and Plant Productivity. Aca- cytoplasm by the 3-P-glycerate/dihydroxyacetone phosphate shut- demic Press, New York