Malate Formation Research Articles

Laboratory tests on glycosidase preparations in wine

The secondary glycosidasic activity of pectinase preparations is used to release flavour active compounds of wine. In order to identify any undesirable effects of other collateral enzymatic activities, laboratory tests were carried out using two commercial preparations: NOVAROM G (Novo-Nordisk) and AR 2000 (Gist-Brocades). A pool of wines was obtained by blending two white wines from neutral grapes (Chardonnay and Verdeca) and a red wine from aromatic grapes (Aleatico). The experiment was carried out by adding each enzymatic preparation to the sample and analysing, by gas chromatography, with flame ionisation and mass spectrometric detection, the free aroma compounds after 15 and 30 days. In the first 15 days, the glycosidasic activity of the NOVAROM G brought about a considerable increase in the wine’s free terpenic content, especially nerol, geraniol and citronellol. After 30 days, further increases in all terpenic compounds were observed. The AR 2000, after 15 days, brought about the release of a conspicuous amount of terpenes, more than the previous enzymatic preparation, which further increased after 30 days. Throughout the test period, the enzymatic preparation, NOVAROM G, had no effect on the content of the acetates of the higher alcohols, the compounds associated with the banana-pineapple (isoamyl acetate) and rose (2-phenyl ethyl acetate) scents. However, the AR 2000 brought about a rapid hydrolysis of total acetates, which caused a fifty percent reduction in isoamyl acetate after 15 days and continued to decline until, after 30 days, the remaining concentration was below the perception threshold. Neither of the enzymatic preparations, however, provoked significant effects on the ethyl esters of the fatty acids compared to the control wine. In addition, the AR 2000 revealed esterase activity involving fixed acids, such as malic acid and succinic acid, with a conspicuous formation of diethyl malate and diethyl succinate, which became more marked after 30 days.

Isolation of High-Malate-Producing Sake Yeasts from Low-Maltose-Assimilating Mutants.

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.

Isolation of high-malate-producing sake yeasts from low-maltose-assimilating mutants

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.

Oxidation–Reduction Properties of Two Engineered Redox-Sensitive Mutant Escherichia coli Malate Dehydrogenases

Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The Em is −285 mV when cysteines are at positions 121 and 305 and −295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects kcat but not Km values. In the single V121C and N122C mutants, the Cys in helix-5 affects the Km for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.

Transgenic potato plants with altered expression levels of chloroplast NADP-malate dehydrogenase: interactions between photosynthetic electron transport and malate metabolism in leaves and in isolated intact chloroplasts

The contribution of the malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Desiree) plants with altered expression of plastidic NADP-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial.

Role of phosphoenolpyruvate carboxylase in malate production by the developing barley aleurone layer

The pathway of malate synthesis in the developing aleurone layer of barley (Hordeum vulgare L. cv. Himalaya) was investigated. Malate formation did not occur under anoxia. Labelling with [2‐14C]acetate showed that the glyoxylate pathway was not a significant source of malate. The partitioning of glycolytic carbon flux at the branchpoint between phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) and pyruvate kinase (PK, EC 2.7.1.40) was studied using [U‐14C]glucose. It was concluded that in aleurone from maturing, rapidly acidifying grains the flux through the PEPC branch relative to that through PK is 3‐5 times greater than in young aleurone. This increase in flux can be accounted for by a 5‐fold increase in PEPC protein determined by western blotting and in PEPC activity measured in vitro.

Flux Control of the Malate Valve in Leaf Cells

The coupled processes of the chloroplast trans-envelope transport of malate and oxaloacetate and their interconversion as catalyzed by the stromal NADP-linked malate dehydrogenase are quantitatively analyzed by means of a steady-state model. The equation for the NADP–malate dehydrogenase reaction is developed. The empirical dependence of enzyme activity on NADPH and NADP+is used to determine its actual activity. The trans-envelope counter exchange of malate and oxaloacetate is described by a kinetic model of the translocator. Kinetic parameters are derived from known data, except for theKmvalue and the maximum rate for oxaloacetate transport, which are estimated from oxaloacetate-dependent malate formation in isolated intact chloroplasts. Using the kinetic properties of the system and the known metabolite concentrations, the model demonstrates that photosynthetically generated NADPH can be exported efficiently from the chloroplasts to the cytosol by the malate-valve system. The transfer capacity of the malate valve is estimated not to exceed 20 μmol (mg Chl)−1h−1(or 5% of the electron transport) under normal physiological conditions. The possible role of the malate valve in leaf cells under normal conditions and during stress is discussed.

Competition between electron acceptors in photosynthesis: Regulation of the malate valve during CO2 fixation and nitrite reduction

For maximal rates of CO2 assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO2 fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO2 fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO2 fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the 'malate valve' prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The 'malate valve' is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the 'malate valve' functions at various redox states of the acceptor side of Photosystem I.

The absence of glyxylate cycle enzymes in rodent and embryonic chick liver

There have been several reports over the past decade of the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase in animal tissues. Reaction products in these assays have been measured principally by chromatographic separation of isotopes or by colorimetric procedures. In this report more sensitive and accurate HPLC and HPCE analyses were used to detect enzymatic activity. Reversed phase HPLC revealed the absence of detectable isocitrate lyase activity in guinea pig, rat and chick embryonic liver. The formation of several other α-keto acids was detected and this may account for the previously reported activities. Using HPCE to monitor malate formation malate synthase activity was not detected in these tissues. These results indicate that when assaying enzyme activities in crude tissue homogenates specific methods for the identification of end products are required.

Differential effects of drought and light levels on accumulation of citric and malic acids during CAM in Clusia

ABSTRACTNight‐time citrate accumulation has been proposed as a response to stress in CAM plants. To address this hypothesis, gas exchange patterns and nocturnal acid accumulation in three species of Clusia were investigated under controlled conditions with regard to water stress and responses to low and high photosynthetic photon flux density (PPFD). Under high PPFD, leaves of Clusia nocturnally accumulated large amounts of both malic and citric acids. Under low PPFD and well‐watered conditions, substantial night‐time citrate accumulation persisted, whereas malate accumulation was close to zero. Malate accumulation and night‐time CO2 uptake from the atmosphere declined in all three species during prolonged drought periods, whereas citrate accumulation remained similar or increased. Recycling of respiratory CO2 was substantial for both well‐watered and water‐stressed plants. The suggestion that citrate accumulation is energetically more favourable than malate accumulation is not supported if the source of CO2 for the formation of malate is respiratory CO2. However, the breakdown of citric acid to pyruvate in the light period releases three molecules of CO2, while the breakdown of malic acid releases only one CO2 per pyruvate formed. Thus, citric acid should be more effective than malic acid as a mechanism to increase CO2 concentration in the mesophyll and may help to prevent photoinhibition. Organic acid accumulation also affected the vacuolar pH, which reached values of 2·6–3·0 at dawn. At these pH values, the transport of 2H+/ATP is still feasible, suggesting that it is the divalent form of citrate which is being transported in the vacuoles. Since citrate is a well‐known buffer, and Clusia spp. show the largest day‐night changes in organic acid levels measured in any CAM plant, it is possible that citrate increases the buffer capacity of the vacuoles. Indeed, malate and titratable acidity levels are positively related to citrate levels. Moreover, Clusia species that show the highest nocturnal accumulation of organic acids are also the ones that show the greatest changes in citric acid levels.

Malate Metabolism in the Dark After 13CO2 Fixation in the Crassulacean Plant Kalanchoë tubiflora

The metabolism of [(13)C]malate was studied in the Crassulacean plant Kalanchoë tubiflora following exposure to (13)CO(2) for 2 hour intervals during a 16 hour dark cycle. Nuclear magnetic resonance spectroscopy of [(13)C]malate extracted from labeled tissue revealed that the transient flux of malate to the mitochondria, estimated by the randomization of [4-(13)C]malate to [1- (13)C]malate by fumarase, varied substantially during the dark period. At both 15 and 25 degrees C, the extent of malate label randomization in the mitochondria was greatest during the early and late parts of the dark period and was least during the middle of the night, when the rate of (13)CO(2) uptake was highest. Randomization of labeled malate continued for many hours after malate synthesis had initially occurred. Internally respired (12)CO(2) also served as a source of carbon for malate formation. At 15 degrees C, 15% of the total malate was formed from respired (12)CO(2), while at 25 degrees C, 49% of the accumulated malate was derived from respired (12)CO(2). Some of the malate synthesized from external (13)CO(2) was also respired during the night. The proportion of the total [(13)C]malate respired during the dark period was similar at 15 and 25 degrees C, and respiration of newly formed [(13)C]malate increased as the night period progressed. These data are discussed with regard to the relative fluxes of malate to the mitochondria and the vacuole during dark CO(2) fixation.

Characterization of reactions catalyzed by manganese peroxidase from Phanerochaete chrysosporium

Manganese peroxidase (MnP) is one of two extracellular peroxidases believed to be involved in lignin bio-degradation by the white-rot basidiomycete Phanerochaete chrysosporium. The enzyme oxidizes Mn(II) to Mn(III), which accumulates in the presence of Mn(III) stabilizing ligands. The Mn(III) complex in turn can oxidize a variety of organic substrates. The stoichiometry of Mn(III) complex formed per hydrogen peroxide consumed approaches 2:1 as enzyme concentration increases at a fixed concentration of peroxide or as peroxide concentration decreases at a fixed enzyme concentration. Reduced stoichiometry below 2:1 is shown to be due to Mn(III) complex decomposition by hydrogen peroxide. Reaction of Mn(III) with peroxide is catalyzed by Cu(II), which explains an apparent inhibition of MnP by Cu(II). The net decomposition of hydrogen peroxide to form molecular oxygen also appears to be the only observable reaction in buffers that do not serve as Mn(III) stabilizing ligands. The nonproductive decomposition of both Mn(III) and peroxide is an important finding with implications for proposed in vitro uses of the enzyme and for its role in lignin degradation. Steady-state kinetics of Mn(III) tartrate and Mn(III) malate formation by the enzyme are also described in this paper, with results largely corroborating earlier findings by others. Based on a comparison of pH effects on the kinetics of enzymatic Mn(III) tartrate and Mn(III) malate formation, it appears that pH effects are not due to ionizations of the Mn(III) complexing ligand.

Carbon flux through tricarboxylic acid cycle in rat renal tubules

Our aim was to delineate the effect(s) of chronic metabolic acidosis on renal TCA-cycle metabolism. Renal tubules isolated from control and chronically acidotic rats were incubated at pH 7.4 with either 2 mM [2,3- 13C]pyruvate or [2- 13C]acetate. GC-MS and/or 13C-NMR were utilized to monitor the flux of 13C through pyruvate dehydrogenase, pyruvate carboxylase and the TCA-cycle. With either, precursor acidosis was associated with significantly decreased formation of 13C-labelled citrate, malate, aspartate and alanine and increase formation of glucose, lactate and acetyl-CoA as compared with the control. The results indicate that adaptation of renal metabolism to chronic metabolic acidosis is associated with diminished flux through citrate synthetase and concomitantly increased flux through pyruvate carboxylase. The data suggest that depletion of TCA-cycle intermediates and enhanced ammoniagenesis in the kidney of chronically acidotic rats may be regulated at the site of mitochondrial citrate-condensing enzyme.

Studies on the anaerobic energy metabolism in the foot muscle of marine gastropod Patella caerulea (L.)

1. 1. The foot muscle of P. caerulea has a complete sequence of glycolytic enzymes. The low activitiy of hexokinase, in comparison with the activities of glycogen phosphorylase and phosphofructokinase, indicate that glycogen is the main fuel oxidized. 2. 2. The reduction of aspartate content in combination with the accumulation of alanine and the presence of considerable activities of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase indicates a coupled metabolism of glycogen and aspartate during exposure to air. 3. 3. From the changes in the concentration of the metabolites during exposure to air it appears that up to the second hour of anaerobiosis alanine, lactate and glutamate are the end-products which accumulate in the foot muscle of P. caerulea, whereas from the second to the fourth hour only succinte and alanine accumulate. 4. 4. The low activities of the Krebs cycle enzymes as well as the absence of α-ketoglutarate dehydrogenase activity suggest that the Krebs cycle is not in operation. 5. 5. The absence of opine dehydrogenases shows that the end products octopine, alanopine and strombine are not accumulated in the foot muscle under anaerobiosis. 6. 6. The high activity of malate dehydrogenase in the direction of malate formation in combination with the low activity of d-lactate dehydrogenase and the absence of opine dehydrogenases suggests that the former dehydrogenase is coupled 1:1 to glyceraldehyde-3-phosphate dehydrogenase.

Control of pyruvate carboxylase activity by the pyridine-nucleotide redox state in mitochondria from rat liver.

Pyruvate carboxylation by isolated mitochondria from rat liver is inhibited by t-butylhydroperoxide in a fully reversible manner. The rate of malate formation at 10 mM pyruvate was decreased by some 80% by 30 microM t-butylhydroperoxide. The effective peroxide concentration was dependent on the mitochondrial hydrogen supply, being increased to about 120 microM in the presence of 50 microM palmitoylcarnitine. Regarding the mechanism(s) of the t-butylhydroperoxide action, pyruvate transport and intramitochondrial energy or activator supply are unlikely involved, because the effect also took place with alanine as the substrate and was not accompanied by a change in the intramitochondrial levels of adenine nucleotides and acetyl-CoA respectively. However, t-butylhydroperoxide caused a rapid fall in the 3-hydroxybutyrate/acetoacetate ratio and a marked increase in the oxidized glutathione content. Therefore, experiments were designed to disclose the participation of the respective redox couples in the expression of pyruvate carboxylase activity. From measurements of NADPH, NADH, oxidized and reduced glutathione contents of mitochondria incubated under a variety of conditions, evidence has been obtained indicating that the mitochondrial NADH supply represents an important factor in the regulation of pyruvate carboxylase activity. The results presented seemingly provide a new basis for the understanding of the functional relationship between beta-oxidation and pyruvate carboxylation.

Presence of anaplerotic reactions and transamination, and the absence of the tricarboxylic acid cycle in mollicutes.

Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added 14CO2 led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.

Differences in intermediary energy metabolism between juvenile and adult Fasciola hepatica

A comparison of glucose catabolism by juvenile and adult liver flukes, Fasciola hepatica, showed that in the adult the cytosolic degradation of glucose via phosphoenolpyruvate carboxykinase (PEPCK) was the most important route, whereas in the freshly excysted juvenile a large part was degraded via pyruvate kinase (PK). However, it was also shown that the adult did not exclusively use the PEPCK pathway, nor did the juvenile exclusively use the PK pathway. When the juvenile was forced to anaerobic functioning it produced propionate and acetate just like the adult, but this did not imply that it switched to the pathways of the adult: the pathway via PK remained important. Malic enzyme (NADP(H)-dependent) was demonstrated to be present in the cytosol and in the mitochondria of both juveniles and adults. These enzyme activities enable the parasite to use a mixture of malate and pyruvate in any ratio as substrate for the mitochondrial production of proportionate and acetate. Pyruvate dismutation was important in the anaerobically functioning juvenile, whereas in the adult malate was the major, but not the only mitochondrial substrate. The pH profiles of PK and PEPCK showed that the pathway of PEP metabolism at the PK/PEPCK branchpoint can be regulated by the pH. However, the end products of glucose breakdown were not dependent on the pH. During its development, the liver fluke will gradually be forced to anaerobic functioning. At first, the acidic end product will favour a partitioning of PEP at the PK/PEPCK branchpoint toward malate formation. Later, a lasting predominance of the PEPCK pathway occurs as PK activity almost completely disappears. During the development of F. hepatica also a transition in the mitochondria occurs: at the acetyl-coenzyme A branchpoint a switch from citrate to acetate formation could be demonstrated. The most likely mechanism of this change is discussed.

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

1. 1. The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (μmol/min)/mg at 30°C. 2. 2. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. 3. 3. The K m values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 μM and 13.5 mM, respectively. The K m values for malate, NADP and Mn 2+ in the decarboxylation reaction were 0.1 mM, 25 μM and 5 μM, respectively. 4. 4. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. 5. 5. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.

NADP-malate Dehydrogenase Activity during Photosynthesis in Illuminated Spinach Chloroplasts

Summary Light-dependent O 2 -evolution, redox state of NADP(H), activation state of the NADP-malate dehydrogenase and redox state of thioredoxin m were measured simultaneously in isolated intact spinach chloroplasts exposed to various conditions: CO 2 , 3-phosphoglycerate, oxaloacetate or no electron acceptor were present in the incubation medium. When no reoxidation of the light-generated NADPH could take place because of a lack of electron acceptors, full activation of NADP-malate dehydrogenase as well as reduction of thioredoxin m was observed. However, in the presence of electron acceptors the NADP level remained as high as in the dark and NADP-malate dehydrogenase activation as well as thioredoxin m -reduction appeared largely to be prevented. When chloroplasts were allowed to perform oxaloacetate-dependent O 2 -evolution, the flux through the electron transport chain as indicated by the rate of O 2 -evolution, and the rate of malate formation corresponded exactly to the in situ activation state of the NADP-malate dehydrogenase. We conclude that the fine regulation of the reductive activation of NADP-malate dehydrogenase by NADP (Scheibe and Jacquot, 1983) determines the actual extent of export of reducing equivalents, in the form of malate, via the dicarboxylic acid translocator. Malate production and translocation into the cytoplasm is largely prevented when a high demand for NADPH inside the chloroplast leads to a high NADP/NADPH ratio.