Malate Metabolism Research Articles

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

Regulation of malate dehydrogenases from neonatal, adolescent, and mature rat brain

Since the malate-aspartate shuttle in brain has been shown to be closely linked to brain energy metabolism and neurotransmitter synthesis, the activity of MDH, one of the enzymes of the malate-aspartate shuttle, was studied in cortical non-synaptic mitochondria (mMDH) and cytosol (cMDH) in 1-4 day, 18-20 day and 7-8 week old rats. The mean mMDH activity (nmol/min/mg protein) was 10,517 +/- 734 (mean +/- SEM), 8,882 +/- 241 and 10,323 +/- 561 and cMDH activity was 2,453 +/- 99, 4,673 +/- 152 and 6,821 +/- 205 in 1-4 day, 18-20 day and 7-8 week old rats, respectively. While cMDH activity increased with age (p < 0.0001), mMDH activity showed no change. This study also determined if endogenous compounds, previously shown to alter malate metabolism, affected MDH activities. Lactate inhibited only cMDH activity, by a competitive mechanism. Oxaloacetate inhibited mMDH by partial non-competitive inhibition and cMDH by competitive inhibition. Alpha-ketoglutarate competitively inhibited both enzymes; however, the inhibition of mMDH activity was more pronounced than that of cMDH activity. Citrate inhibited mMDH via an uncompetitive mechanism and cMDH via a noncompetitive mechanism. The mechanisms of inhibition of mMDH and cMDH by each of the effectors were the same over the three ages. The results suggest mMDH and cMDH activities show a dissimilar developmental pattern and may be regulated differently by endogenous effectors. The greater sensitivity of mMDH, compared to cMDH, to certain effectors may be related to the dual role of mMDH in the tricarboxylic acid cycle and the malate-aspartate shuttle.

Uptake and metabolism of malate in neurons and astrocytes in primary cultures.

Uptake and oxidative metabolism of [14C]malate as well as its incorporation into aspartate, glutamate, glutamine, and GABA were studied in cultured cerebral cortical neurons (GABAergic), cerebellar granule neurons (glutamatergic), and cerebral cortical astrocytes. All cell types exhibited high affinity uptake of malate (Km 10-85 microM) with slightly higher Vmax values in neurons (0.1-0.2 nmol x min-1 x mg-1) than in astrocytes (0.06 nmol x min-1 x mg-1). Malate was oxidatively metabolized in all three cell types with nominal rates of 14CO2 production of 2-15 pmol x min-1 x mg-1. The oxidation of malate was only slightly inhibited by 5 mM aminooxyacetic acid (AOAA). In granule cell preparations [14C]malate was incorporated into aspartate and glutamate and, to a much less extent, into glutamine. This incorporation was blocked by 5 mM AOAA. Astrocytes exhibited slightly higher incorporation rates into aspartate and glutamate, but in these cells glutamine was labelled to a considerable extent. AOAA (5 mM) inhibited the incorporation by 60-70%. In cultures of cerebral cortical neurons, very low levels of radioactivity derived from [14C]malate were found in aspartate and glutamate, and GABA was not labelled at all. Glutamine had the same specific activity as glutamate, indicating that the low rates of incorporation of radioactivity into amino acids in this preparation is likely to exclusively represent metabolism of malate in the small population of astrocytes (5% of total cell number), contaminating the neuronal cultures. The findings suggest that exogenous malate to a quantitatively limited extent may serve as a precursor for transmitter glutamate in glutamatergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Conversion of Malate and Localization of Enzymes Involved in the Metabolism of Malate in Photoautotrophic Cell Cultures of Chenopodium rubrum

Cells from photoautotrophic cultures of Chenopodium rubrum were fractionated for the isolation of purified chloroplasts and mitochondria. The subcellular localization of the enzymatic activities involved in the metabolism of malic acid was investigated. Highly purified chloroplasts were obtained from the protoplasts, whereas peroxisomes were still present in the mitochondrial fraction. NAD - and NADP-dependent malate dehydrogenase and malic enzyme activities were found in the mitochondrial and chloroplast fractions, respectively. Exogenously supplied [14C]labelled malate was metabolized by the photoautotrophic cell suspension, obviously to the greater part in mitochondria.

Tansley Review No. 37 Circadian rhythms: their origin and control.

This article reviews the circadian rhythm of carbon dioxide metabolism in leaves of the Crassulacean plant Bryophyllum (Kalanchoë) fedtsckenkoi which persists both in continuous darkness and a CO2 -free atmosphere, and in continuous light and normal air. Under both conditions the rhythm is due to the periodic activity of the enzyme phosphoenolpyruvate carboxylase (PEPc). The physiological characteristics of the rhythm are described in detail and, from these characteristics, hypotheses are advanced to account for both the generation of the rhythm and the regulation of its phase and period by environmental factors. The periodic activity of PEPc is ascribed to the periodic accumulation of an allosteric inhibitor, malate, in the cytoplasm and its subsequent removal either to the vacuole in continuous darkness, or by metabolism in continuous light. Also involved in the generation of the rhythm is a periodic change in the sensitivity of PEPc to malate inhibition due to the periodic phosphorylation and dephosphorylation of PEPc which changes its K1 by a factor of 10 from 30 to 0.3 mM and vice versa. This periodic phosphorylation of PEPc is apparently achieved by the periodic synthesis and breakdown of a PEPc kinase which phosphorylates the enzyme on a serine residue; dephosphorylation is achieved by a type 2A phosphatase which shows no rhythmic variation. The induction of phase shifts in the rhythm in continuous darkness and CO2 -free air has been explained in terms of light and high-temperature activated gates or channels in the tonoplast which, when open, allow malate to diffuse between the vacuole and cytoplasm. For the rhythm in continuous light and normal air phase, control by environmental signals can be attributed to changes in the malate levels in critical cell compartments, or in particular cell populations such as the stomatal guard cells, due to regulation of the malate synthesizing enzyme system involving PEPc, and malic enzyme which is responsible for malate metabolism. The role of the stomata in the generation of the rhythm is also discussed. The biochemical events which appear to give rise to the well-studied circadian rhythms in leaf movement in Samanea and Albizza, in luminescence in Gonyaulax polyedra and in the synthesis of the chlorophyll a/b binding protein are also reviewed in an attempt to identify similarities between these events and those involved in the Bryophyllum rhythm. Finally, the somewhat similar nature of the genes apparently responsible for circadian rhythmicity in Neurospora and Drosophila are discussed, and suggestions made for utilizing anti-sense nucleic acid technology in the further elucidation of the critical biochemical events involved in the basic, temperature-compensated circadian oscillator in living organisms. CONTENTS Summary 347 I. Introduction 348 II. Occurrence of circadian rhythms 348 III. Physiological characteristics of circadian rhythms 349 IV. Biochemical and molecular events involved in the circadian rhythm in Bryophyllum leaves 362 V. Biochemical and molecular events involved in the origin and control of circadian rhythmicity in other organisms 366 VI. Genetic studies 370 VII. Conclusion 371 References 372.

Effect of nitrate limitation on the photosynthetically active pools of aspartate and malate in maize, a NADP malic enzyme C4 plant

After two weeks of moderate N restriction, growth of 3‐week‐old Zea mays L. plants was less than half that of the control and aspartate and malate levels in the leaves were severely suppressed (45 and 65% decrease, respectively). Since in NADP malic enzyme type C4 plants, such as maize, malate and aspartate are intermediates in the C4 photosynthetic pathway, the operation of the latter was investigated. Moderate nitrogen deficiency had only a small effect on the rate of photosynthesis (20% decrease) measured under 1000 umol m−2 s−1 irradiance. 14CO2 pulse‐12CO2 chase experiments combined with measurements of in vitro photosynthetic enzyme activities demonstrated the operation of a typical C4 photosynthetic pathway in N‐restricted plants. The turnover rates of malate and aspartate molecules involved in the C4 cycle were determined by the loss of label in the carbon 4 moiety of these molecules during the chase period. It is shown that N restriction did not alter the turnover of malate but greatly accelerated that of aspartate. The amounts of malate and aspartate moving through photosynthetically active pools were estimated using a kinetic model. For malate, the size of this pool appeared to be only slightly diminished whereas for aspartate the size of the corresponding pool decreased by a factor of 3. It is proposed that under moderate NO3− deficiency, despite deviations in malate metabolism leading to a pronounced decrease in the size of its cellular pool, a large amount of malate remained in the operation of the C4 pathway. By contrast, the participation of aspartate in the operation of the C4 pathway was greatly reduced.

Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy

(31)P-, (13)C-, and (15)N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and gamma-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gln, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from Glc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.

Cytoplasmic malate levels in maize root tips during K + ion uptake determined by 13C-NMR spectroscopy

13C-NMR spectroscopy was used to determine the level of cytoplasmic malate in maize root tips that exhibited different rates of malate synthesis. Intracellular malate was 13C-labeled at carbons 1 and 4 by perfusing root tips with 5 mM H 13CO 3 −. This labeling reflects the activities of phospho enolpyruvate carboxylase and malate dehydrogenase (production of [4- 13C]malate), and fumarase (scrambling of 13C-label between C1 and C4 of malate). In vivo 13C-NMR spectra contained a clearly resolved resonance from cytoplasmic [4- 13C]malate, while the resonance from cytoplasmic [1- 13C]malate overlapped with others. After 90 min of H 13CO 3 − treatment, 13C-labeling of organic acid pools and reached steady-state. Thereafter, the ratios [ 13 C] malate/[ 12 C + 13 C] malate and [1- 13C]malate/[4- 13C]malate in tissue extracts remained constant; evidence is presented that these ratios were the same for both cytoplasmic and total cellular malate. Hence, the intensity of the cytoplasmic [4- 13C]malate signal was proportional to the amount of cytoplasmic malate in root tips. Potassium sulfate stimulated malate synthesis in maize root tips, relative to root tips perfused with HCO 3 − alone; total cellular malate doubled after approx. 1 h of 5 mM K 2SO 4-treatment. Cytoplasmic malate increased from approx. 3.5 mM to approx. 7.5 mM within 45 min of the onset of K 2SO 4-treatment, declining slightly thereafter. The possible effects of these changing cytoplasmic malate concentrations on the enzymes involved in malate metabolism are discussed.

Hepatocyte heterogeneity in uptake and metabolism of malate and related dicarboxylates in perfused rat liver

1. In isolated perfused rat liver a near-maximal net malate uptake of about 120 nmol g-1 min-1 was observed at influent malate concentrations above 100 mumol l-1 and a half-maximal uptake at about 50 mumol l-1 in influent. 14CO2 production from added [U-14C]malate paralleled hepatic net malate uptake, however, 14CO2 production exceeded net malate uptake by 20-25%. This was observed in antegrade as well as in retrograde perfusions and regardless of whether NH4Cl was added to the influent perfusate. Stimulation of glutamine synthesis by NH4Cl only slightly affected net malate uptake and 14CO2 production, but resulted in a marked stimulation of [14C]glutamine release from the liver. 2. Because [U-14C]malate uptake by the liver (reflecting the influent/effluent concentration difference of labeled malate) could at least in part involve a malate/malate exchange mechanism, net malate uptake (as determined from the influent/effluent concentration difference of enzymatically assayable malate) may underestimate hepatic [U-14C]malate uptake. On the other hand, during metabolic steady states 14CO2 production from added [U-14C]malate can be considered as an upper limit estimate of [U-14C]malate uptake by the liver. Assuming that 14CO2 production equals [U-14C]malate uptake by the liver, extrapolation studies suggest that during maximal rates of NH4Cl-stimulated glutamine synthesis 80-110% of the [U-14]malate taken up by the liver was used for glutamine synthesis. This was true for retrograde and antegrade perfusions. Similar data, i.e. a 100-130% incorporation regardless of the direction of perfusion, were obtained when [U-14C]malate uptake was assumed to equal net malate uptake by the liver. 3. Substitution of Na+ in the perfusion fluid by choline abolished net malate uptake by the liver and inhibited 14CO2 production from [U-14C]malate by more than 90%. 4. 2-Oxoglutarate inhibited [14C]malate uptake and [1-14C]oxoglutarate uptake by the liver was inhibited by malate, fumarate, succinate and oxaloacetate, but not by aspartate and glutamate. Inhibition of [1-14C]oxoglutarate uptake and of 14CO2 production from added labeled 2-oxoglutarate by malate and fumarate seemed largely competitive. Malate, fumarate and succinate not only inhibited [1-14C]oxoglutarate uptake, but also stimulated the release of unlabeled 2-oxoglutarate from the liver. 5. The data are consistent with a predominant uptake of vascular malate by perivenous glutamine synthetase containing hepatocytes when glutamine synthesis is stimulated to Vmax values by NH4Cl. Malate and other citric acid cycle dicarboxylates, but not aspartate and glutamate, may compete with 2-oxoglutarate for uptake into perivenous glutamine synthesizing hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

The metabolism of malate by cultured rat brain astrocytes

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-[U-14C]malate in primary cultures of rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from 0.01 mM and 0.5 mM malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain. Both the biphasic kinetics and the differential action of many of the effectors on the 14CO2 production from 0.01 mM and 0.5 mM malate provide evidence for the presence of more than one pool of malate metabolism in cultured rat brain astrocytes.

Detection of the monocarboxylate transporter from pea mitochondria by means of a specific monoclonal antibody

The monocarboxylate (pyruvate) transporter from pea ( Pisum sativum) mitochondria was identified by means of a specific monoclonal antibody. The antibody blocked pyruvate-dependent oxaloacetate metabolism without interfering with the metabolism of malate, α-ketoglutarate, or glycine. The antibody also blocked the pyruvate/pyruvate exchange reaction of the partially purified transporter reconstituted into phospholipid membranes. Using the specific monoclonal antibody, the transporter was identified on Western blots as a minor 19 kDa protein.

Tissue specificity of the mitochondrial forms of malic enzyme in herring tissues

Abstract 1. 1. The activity of malic enzyme per wet weight or per milligram of mitochondrial protein in herring tissues showed significant tissue specificity. 2. 2. The specific activity of herring malic enzyme per mg of mitochondria from testes was 30 times greater than that of malic enzyme found in mitochondria of ovaries. 3. 3. The NAD(P)-dependent mitochondrial malic enzyme was present in all tissues tested and testis mitochondria contained the highest activity of this molecular form. 4. 4. Herring skeletal muscle mitochondria contained nearly identical activities of both molecular forms, the NADP- and NAD(P)-dependent malic enzyme, respectively. 5. 5. The results extend and support the proposal that mitochondria of some fish may contain two molecular forms of malic enzyme and have a unique intramitochondrial pahtway for malate metabolism.

Phase resetting of the circadian rhythm of carbon dioxide assimilation inBryophyllum leaves in relation to their malate content following brief exposure to high and low temperatures, darkness and 5% carbon dioxide

Leaves ofBryophyllum fedtschenkoi show a persistent circadian rhythm in CO2 assimilation when kept in continuous illumination and normal air at 15°C. The induction of phase shifts in this rhythm by exposing the leaves for four hours at different times in the circadian cycle to 40° C, 2° C, darkness and 5% CO2 have been investigated. Exposure to high temperature has no effect on the phase at the apex of the peak but is effective at all other times in the cycle, whereas exposure to low temperature, darkness or 5% CO2 is without effect between the peaks and induces a phase shift at all other times. The next peak of the rhythm occurs 17 h after a 40° C treatment and 7-10 h after a 2° C, dark or 5% CO2 treatment regardless of their position in the cycle. When these treatments are given at times in the cycle when they induce maximum phase shifts, they cause no change in the gross malate status of the leaf. The gross malate content of the leaf in continuous light and normal air at 15% shows a heavily damped circadian oscillation which virtually disappears by the time of the third cycle, but the CO2 assimilation rhythm persists for many days. The generation of the rhythm, and the control of its phase by environmental factors are discussed in terms of mechanisms that involve the synthesis and metabolism of malate in specific localised pools in the cytoplasm of the leaf cells.

Malic Enzyme Activity in Bacteroids from Soybean Nodules

Soluble extracts of Bradyrhizobium japonicum bacteroids from soybean root nodules showed substantial rates of NAD+ and NADP+ reduction which were malate and MnCl2 dependent. Pyruvate was formed stoichiometrically and the NAD- and NADP-dependent rates were additive, indicating the presence of two malic enzymes. The NADP-dependent malic enzyme had a high affinity for malate (apparent K m = 0·1 mm) and was stimulated by ammonium. The NAD-dependent malic enzyme had a lower affinity for malate (apparent K m = 1·9 mM) and was stimulated by potassium and ammonium salts. The maximum velocities of the two enzymes were similar and of comparable magnitude to the activities of tricarboxylic acid cycle enzymes in the extracts. Possible roles of the malic enzymes in the metabolism of malate and succinate in bacteroids are discussed.

Enzymes of carbohydrate metabolism ofCotugnia digonopora and their activity in the presence of anthelmintics,in vitro

Cotugnia digonopora, a fowl cycllophyllidean cestode, was found to possess most of the enzymes, associated with the glycolytic sequence and phosphoenolpyruvate branch point, in the cytosol fraction. Enzymes of malate metabolism were predominantly mitrochondrial. Anthelmintic agents inhibited hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, fumarate reductase, and malic enzyme. In intact worms this effect was significantly reduced. However, the activities of glycogen Phosphorylase and pyruvate kinase were significantly enhanced.

The renal toxicity of ( R)-3-chlorolactate in the rat

The renal toxicity of ( R, S)-3-chlorolactate has been shown to be due to the ( R)-isomer which, when administered to rats, induces diuresis and glucosuria. The metabolic activity of isolated tubule cells, prepared from rat kidney, was inhibited by ( R)-3-chlorolactate and the action of the compound was localised as affecting mitochondrial metabolism. Studies with kidney mitochondria pin-pointed the site of action as being involved with the oxidative metabolism of malate but not the inhibition of mitochondrial malate dehydrogenase. The effects of oxalate, a metabolite of ( R)-3-chlorolactate, and of ( R, S)-3-chlorolactaldehyde on renal tubule cells was investigated. While some degrees of inhibition of metabolic activity were evident, these compounds were not responsible for the toxic effects produced by ( R)-3-chlorolactate.

The role of malate in ammonia assimilation in cotyledons of radish (Raphanus sativus L.)

The relationships between the metabolism of malate, nitrogen assimilation and biosynthesis of amino acids in response to different nitrogen sources (nitrate and ammonium) have been examined in cotyledons of radish (Raphanus sativus L.). Measurements of the activities of some key enzymes and pulse-chase experiments with [(14)C]malate indicate the operation of an anaplerotic pathway for malate, which is involved in the synthesis of glutamine during increased ammonia assimilation. It is most likely that the tricarboxylicacid cycle is supplied with carbon through entry of malate, formed via the phosphoenolpyruvate (PEP)-carboxylation pathway, when 2-oxoglutarate leaves the cycle to serve as precursor for an increased synthesis of glutamine via glutamate. This might occur predominantly in the cytosol via the activity of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, the NADH-dependent GOGAT being the rate-limiting activity.

Evidence for a relationship between malate metabolism and activity of 1-sinapoylglucose: L-malate sinapoyltransferase in radish (Raphanus sativus L.) cotyledons

The control of malate metabolism and stimulation of 1-sinapolyglucose: L-malate sinapoyltransferase (SMT) activity in radish (Raphanus sativus L. var. sativus) cotyledons has been studied. The light-induced and nitrate-dependent activity of SMT catalyzes the formation of O-sinapoly-L-malate via 1-O-sinapoyl-β-D-glucose. When dark-grown radish seedlings, cultivated in quartz sand with nutrient solution containing NO 3 (-) as the sole N source, were treated with light, SMT activity increased concomitantly with free malate in the cotyledons. This light effect was suppressed in seedlings grown in a culture medium which contained in addition to NO 3 (-) also NH 4 (+) . However, treatment with methionine sulfoximine neutralized this ammonium effect, resulting again in both rapid accumulation of malate and rapid increase in SMT activity. When seedlings grown on NO 3 (-) nitrogen were subsequently supplied with NH 4 (+) nitrogen, the accumulated level of L-malate rapidly dropped and the SMT increase ceased. The enzyme activity decreased later on, reaching the low activity level of plants which were grown permanently on NO 3 (-) /NH 4 (+) -nitrogen. An external supply (vacuum infiltration) of malate to excised cotyledons and intact seedings, grown on NO 3 (-) /NH 4 (+) -nitrogen medium, specifically promoted a dose-dependent increase in the activity of SMT. In summary these results provide evidence indicating that the SMT activity in cotyledons of Raphanus sativus might be related to the metabolism of malic acid.

NON‐PHOTOSYNTHETIC FIXATION OF CARBON DIOXIDE AND POSSIBLE BIOLOGICAL ROLES IN HIGHER PLANTS

Summary1. Non‐photosynthetic fixation of CO2/HCO3‐ occurs both under light and dark conditions and involve the addition of carbon to substrates which in higher plants are derived originally from carbon reduced to carbohydrates during photosynthesis. Despite the endergonic nature of these carboxylations, the advantages offered seem to be sufficient to outweigh the disadvantages of energy loss.2. Non‐photosynthetic carbon incorporation into metabolism is dealt mainly in relation to PEP carboxylase, acetyl‐CoA carboxylase, carbamoyl phosphate synthetase and phosphoribosylaminoimidazole carboxylase while other carboxylases await further characterization or discovery. The extent to which a carboxylase participates depends upon the need for products of its activity in metabolism.3. Non‐photosynthetic carbon fixation is intricately involved in several pathways of metabolism throughout the ontogeny of plants. The roles in relation to leaf carbon metabolism, respiratory metabolism, nitrogen metabolism, lipid and isoprenoid biosynthesis, purine and pyrimidine metabolism and metabolism associated with the action of growth regulators have been described. The fixation reactions appear to be largely concerned with the production of intermediary metabolites, circumvention of energy barriers in metabolism and regulation of plant metabolism. In addition, the activity of PEP carboxylase is involved in ionic balance and pH‐stat.4. Malate derived by way of PEP carboxylase and NAD‐malate dehydrogenase acts as an effective osmoticum and a counter‐ion for K+ accumulation in actively growing plant cells. In addition, malate may enter the TCA cycle or can be decarboxylated by cytoplasmic NADP‐malic enzyme converting NADH to NADPH. Wherever it has been sought in different plant tissues, some evidence for PEP carboxylase and metabolism of malate has always been found.5. Almost every plant process spanning from seed development and germination to flowering and fruit‐set requires the essential participation of non‐photosynthetic carbon fixation in regulating certain metabolic and cellular functions but it does not contribute in a major way to the carbon nutrition of plants. It is largely the tissue type that appears to determine which of the roles is predominant at any one time.

Effect of NAD and Rotenone on the Partitioning of Malate Oxidation Between Malate Dehydrogenase and Malic Enzyme in Isolated Plant Mitochondria

Our aim was to determine whether there is a specific link between NAD-malic enzyme and the rotenone- insensitive bypass of electron transport. Mitochondria were isolated from fresh beetroot tissue, aged beetroot slices, and turnips. Oxygen uptake and pyruvate production were measured in reactions where these mitochondria were metabolizing malate at pH 6.8 in the presence of glutamate, to facilitate the removal of oxaloacetate, and in its absence. In the absence of glutamate there was substantial activity of malic enzyme. NAD+ (577 �M) prevented a fall in oxygen uptake by stimulating malic enzyme. Rotenone (19 �M) reduced oxygen uptake. This inhibited rate was stimulated by NAD+ due, in particular, to a stimulation of malic enzyme. We conclude that the stimulation of malate metabolism by NAD+ is accounted for by malic enzyme due to the unfavourable equilibrium of malate dehydrogenase for malate oxidation and the resultant accumulation of oxaloacetate, and not to any specific link between malic enzyme and the rotenone-insensitive bypass. In the presence of glutamate, malate dehydrogenase was the predominant malate metabolizing enzyme. Oxygen uptake and malic enzyme were stimulated and inhibited by NAD+ and rotenone, respectively. In the presence of rotenone, NAD+ stimulated oxygen uptake and increased the percentage due to malic enzyme. This stimulation is accounted for by the higher Kin of the rotenone-insensitive dehydrogenase for NADH and the unfavourable equilibrium position of malate dehydrogenase resulting in activation of malic enzyme only. We conclude that malic enzyme is not specifically linked to the rotenone-insensitive pathway of electron transport. This has important implications for the regulation of energy metabolism in plants.