mMDH Activity Research Articles

Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana

Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower Ki. Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.

Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling.

Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a (13)CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1 The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and (13)C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions.

Characterization of the Kinetics of Cardiac Cytosolic Malate Dehydrogenase and Comparative Analysis of Cytosolic and Mitochondrial Isoforms

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.

Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

Mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) are sequential enzymes in Krebs cycle. mMDH, CS and the complex between mMDH and CS (mMDH+CS) were treated with nitric oxide solution. The roles of notric oxide (NO) on the secondary structures and the interactions between mMDH and CS were studied using circular diehroism (CD) and Fourier transform surface plasmon resonance (FT-SPR), respectivley. The effects of NO on the activities of mMDH, CS and mMDH+CS were also studied. And the regulations by NO on mMDH and CS were simulated by PyMOL software. The results of SPR confirmed that strong interaction between mMDH and CS existed and NO could significantly regulate the interaction between the two enzymes. NO reduced the mass percents of α-helix and increased that of random in mMDH, CS and mMDH+CS. NO increased the activities of CS and mMDH+CS, and inhibited the activity of mMDH. Graphic simulation indicated that covalent bond was formed between NO and Asn242 in active site of CS. However, there was no direct bond between NO and mMDH. The increase in activity of mMDH+CS complex depended mostly on the interaction between NO and CS. All the results suggested that the regulations by NO on the activity and interaction between mMDH and CS were accord with the changes in mMDH, CS and mMDH+CS caused by NO.

Temperature-dependent alterations in metabolic enzymes and proteins of three ecophysiologically different species of earthworms

The effects of varying temperatures (12 - 44° C) on the specific activity of cytoplasmic malate dehydrogenase ((cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) of some earthworms (Metaphire posthuma, Perionyx sansibaricus and Lampito mauritii) were studied. The effects of different temperatures on supernatant and mitochondrial protein contents were also investigated. The specific activities of cMDH, mMDH and LDH of the earthworms decreased gradually as a function of increasing temperature from 12 to 44°C. Higher metabolic energy was needed to maintain the activity at low temperatures. Hence, the earthworms showed increased enzyme specific activity at low temperatures. However, the protein content increased upto 28°C. Afterwards, with the increase in the temperature from 28 to 42°C, the proteins in the earthworms showed a significant decrease. The temperature-associated changes in the protein content could be explained by the fact that protein synthesizing capacity was hampered above and below the optimum temperature range. The most pronounced effects of varying temperatures were on P. sansibaricus. It might be due to the epigeic nature of the earthworm species. Then minimum effect was on the endogeic earthworm M. posthuma. Virtually, the differences in the enzymes physiology were associated with the differences in the ecological categories of the earthworms. This clearly demonstrate a possible link between the physiology and ecology at aerobic (cMDH, mMDH) and anaerobic (LDH) levels in the tropical earthworms.

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

Malate dehydrogenase (MDH) catalyzes a reversible NAD(+)-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO(2) assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO(2) assimilation/intercellular CO(2) curves at low CO(2), and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.

Alterations in aerobic and anaerobic dehydrogenases and protein levels of three tropical earthworm species with respect to different seasons

The specific activities of cMDH, mMDH and LDH of earthworms (M. posthuma, P. sansibaricus, L. mauritii) started increasing with the onset of favorable seasonal conditions from July to August. A lower temperature and moisture favor the increase in specific activities of enzymes during post-rainy (September–October) and winter (November–January) seasons. The maximum specific activities of enzymes from December to January indicate greater aerobic and anaerobic energy production to cope up with the cold condition. However, the enzyme activities decreased in summer (February–April) with the increase in the ambient temperature. The specific activities were minimum in summer (May–June) as earthworms would be entering quiescent phase to avoid extreme heat and thus showed least energy requirement in this period. Similar seasonal variations were found in the cytoplasmic and mitochondrial proteins. The maximum and minimum seasonal effects were on the epigeic (P. sansibaricus) and endo-anecic (M. posthuma) earthworms, respectively. The differences in the profile of dehydrogenases and proteins may be assigned to the differences in the ecological categories of earthworms.

Habitat-associated changes in some metabolic dehydrogenases and proteins of tropical earthworms

Impacts of different habitats on specific activities of LDH, cMDH, mMDH, cytoplasmic and mitochondrial proteins of M. posthuma, P. sansibaricus, L. mauritii were studied. Maximum specific activity of LDH of the earthworm M. posthuma and L. mauritii was observed in garden. In case of P. sansibaricus, maximum LDH specific activity was found in sewage. Minimum specific activity of LDH of the earthworm M. posthuma was found in non-cultivated field. However, P. sansibaricus and L. mauritii exhibited minimum activity in orchard and cultivated pedoecosystem, respectively. The maximum specific activity of cMDH and mMDH in earthworms from sewage and minimum from non-cultivated field (in case of M. posthuma), garden (in P. sansibaricus) and cultivated land (in L. mauritii) may be due to availability of more organic matter and nitrogen content in the former than in the later cases. Variations in specific activities of these metabolic enzymes due to changes in their pedoecological habitats clearly demonstrate the impact of pedoecosystem on respiratory capacity of the earthworms. Earthworms from pedoecosystem richer in organic matter and nitrogen showed elevated levels of cytoplasmic and mitochondrial protein. The changes in enzyme-specific activity and protein profiles were habitat specific.

Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine

Mitochondrial malate dehydrogenase (mMDH) from the intestine is the NAD-linked oxidoreductase of the tricarboxylic acid cycle with the highest activity and response to vitamin D treatment in vitamin D-deficient chicks (−D). The aim of this study was to elucidate potential molecular mechanisms by which cholecalciferol or calcitriol enhances the activity of this enzyme. One group of animals used was composed of −D and −D treated with cholecalciferol or with calcitriol. A second group consisted of −D and −D supplemented with high Ca 2+ diet. A third group included chicks receiving either a normal or a low Ca 2+ diet. In some experiments, animals were injected with cycloheximide. Data showed that either vitamin D (cholecalciferol or calcitriol) or a low Ca 2+ diet increases mMDH activity. High Ca 2+ diet did not modify the intestinal mMDH activity from −D. The mMDH activity from −D remained unaltered when duodenal cells were exposed to 10 −8 mol/L calcitriol for 15 min. The enhancement of mMDH activity by calcitriol was completely abolished by simultaneous cycloheximide injection to −D. mMDH mRNA levels, detected by RT-PCR, indicate that calcitriol did not affect gene expression. In contrast, Western blots show that calcitriol enhanced the protein expression. In conclusion, calcitriol stimulates intestinal mMDH activity by increasing protein synthesis. No response of mMDH activity by rapid effects of calcitriol or activation through increment of serum Ca 2+ was demonstrated. Consequently, ATP production would be increased, facilitating the Ca 2+ exit from the enterocytes via the Ca 2+-ATPase and Na +/Ca 2+ exchanger, which participate in the intestinal Ca 2+ absorption.

Dictyostelium Differentiation-Inducing Factor-1 Binds to Mitochondrial Malate Dehydrogenase and Inhibits Its Activity

We have reported that the differentiation-inducing factors (DIFs) DIF-1 and DIF-3, morphogens secreted from Dictyostelium discoideum, inhibit proliferation of several cancer cells via suppression of the Wnt/ β-catenin signaling pathway. However, the target molecules of DIFs involved in the anti-proliferative effects are still unknown. In the present study, DIF-1–tethered resins were synthesized to explore the target molecules of DIFs, and mitochondrial malate dehydrogenase (mMDH) was identified as one of the target molecules. In the in vitro assay, DIF-1 and other analogs including 2-MIDIF-1, DIF-3, and 6-MIDIF-3 were found to be capable of binding to mMDH but not to cytoplasmic MDH. However, only DIF-1 and 2-MIDIF-1 inhibited the enzymatic activity of mMDH. The effects of DIF analogs on ATP content and cell proliferation were then analyzed using HeLa cells. DIF-1 and 2-MIDIF-1 were found to lower the ATP content and both chemicals inhibited HeLa cell proliferation, suggesting that inhibition of mMDH activity affected cell energy production, probably leading to the inhibition of proliferation. These results suggest that the inhibition of mMDH activity by DIF-1 and 2-MIDIF-1 could be one of the mechanisms to induce anti-proliferative effects, independent of the inhibition of the Wnt/ β-catenin signaling pathway.

Impact of phorate on malate dehydrogenases, lactate dehydrogenase and proteins of epigeic, anecic and endogeic earthworms

The effects of an organophosphate pesticide phorate on cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH), lactate dehydrogenase (LDH), supernatant and mitochondrial proteins of an epigeic ( Perionyx sansibaricus), anecic ( Lampito mauritii) and endogeic ( Metaphire posthuma) earthworms were studied. The treatment of different concentrations (20, 40, 80 and 160 ppm) of phorate for 16 days gradually decreased the specific activities of cMDH, mMDH and LDH as well as cytoplasmic and mitochondrial protein contents. This showed the inhibitory effect of phorate on metabolic enzymes and proteins in tropical earthworms. The inhibition was dose- and time-dependent. The inhibitory response in mitochondrial enzyme (mMDH) and protein was somewhat earlier and more as compared to the inhibitory effect of phorate on cytoplasmic enzymes (cMDH, LDH) and protein. This indicates a greater interference of phorate in cellular respiration of earthworms. The phorate related decreases in enzyme and protein profiles were about 60% and 58% in P. sansibaricus, 54% and 49% in L. mauritii and 47% and 42% in M. posthuma, respectively. It reflects phorate-induced substantial decline in protein synthesis and aerobic and anaerobic capacity of earthworms. The maximum effect of phorate was on epigeic earthworm followed by anecic and endogeic species. The present findings suggest the differential sensitivity of different earthworm species in enzymatic and protein responses to phorate and the sensitivity was associated with the ecophysiological categories of earthworms.

L‐2‐Hydroxyglutaric aciduria, a defect of metabolite repair

L-2-hydroxyglutaric aciduria is a metabolic disorder in which L-2-hydroxyglutarate accumulates as a result of a deficiency in FAD-linked L-2-hydroxyglutarate dehydrogenase, a mitochondrial enzyme converting L-2-hydroxyglutarate to alpha-ketoglutarate. The origin of the L-2-hydroxyglutarate, which accumulates in this disorder, is presently unknown. The oxidation-reduction potential of the 2-hydroxyglutarate/alpha-ketoglutarate couple is such that L-2-hydroxyglutarate could potentially be produced through the reduction of alpha-ketoglutarate by a NAD- or NADP-linked oxidoreductase. In fractions of rat liver cytosolic extracts that had been chromatographed on an anion exchanger we detected an enzyme reducing alpha-ketoglutarate in the presence of NADH. This enzyme co-purified with cytosolic L-malate dehydrogenase (cMDH) upon further chromatography on Blue Sepharose. Mitochondrial fractions also contained an NADH-linked, 'alpha-ketoglutarate reductase', which similarly co-purified with mitochondrial L-malate dehydrogenase (mMDH). Purified mMDH catalysed the reduction of alpha-ketoglutarate to L-2-hydroxyglutarate with a catalytic efficiency that was about 10(7)-fold lower than that observed with oxaloacetate. For the cytosolic enzyme, this ratio amounted to 10(8), indicating that this enzyme is more specific. Both cMDH and mMDH are highly active in tissues and alpha-ketoglutarate is much more abundant than oxaloacetate and more concentrated in mitochondria than in the cytosol. As a result of this, the weak activity of mMDH on alpha-ketoglutarate is sufficient to account for the amount of L-2-hydroxyglutarate that is excreted by patients deficient in FAD-linked L-2-hydroxyglutarate dehydrogenase. The latter enzyme appears, therefore, to be responsible for a 'metabolite repair' phenomenon and to belong to the expanding class of 'house-cleaning' enzymes.

Endosulfan effects on muscle malate dehydrogenase of the freshwater catfish Clarias batrachus

The effects of a sublethal concentration of an organochlorine pesticide endosulfan were investigated on crude and purified forms of cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH) in the muscle of the freshwater catfish Clarias batrachus. Endosulfan treatment reduced significantly the activity and the specific activity of cMDH and mMDH but had no effect on total cytoplasmic and mitochondrial protein contents. This indicates a decline in the efficiency of aerobic energy metabolism in endosulfan-treated fish. The polyacrylamide gel electrophoresis showed two distinct isoforms (C1 and C2) of cMDH and only one form of mMDH. The inhibition produced by endosulfan in vivo was of mixed noncompetitive/uncompetitive type for crude as well as purified cMDH and mMDH. However, in vitro inhibition was of simple noncompetitive and mixed competitive/noncompetitive type for purified cMDH and mMDH, respectively. Citrate was found to be an uncompetitive inhibitor of cMDH and mixed noncompetitive/uncompetitve inhibitor of mMDH. The inhibitory patterns were modulated by endosulfan. These results demonstrate inhibitory effects of endosulfan on skeletal muscle MDH of the freshwater catfish Clarias batrachus and inhibition effects are mediated through enzyme/substrate/endosulfan complexing.

Role of NADH shuttles in glucose-induced insulin secretion from fetal beta-cells.

The NADH shuttle system, which transports the substrate for oxidative metabolism directly from the cytosol to the mitochondrial electron transport chain, has been shown to be essential for glucose-induced activation of mitochondrial metabolism and insulin secretion in adult beta-cells. We examined the role of these shuttles in the fetal beta-cell, which is immature in being unable to secrete insulin in response to glucose. The activity and concentration of the two key enzymes of the NADH shuttles, mitochondrial glycerol phosphate dehydrogenase (mGPDH) and mitochondrial malate dehydrogenase (mMDH), were eight- and threefold lower, respectively, in fetal compared with adult rat islets. Likewise, mGPDH and mMDH activity was fivefold lower in islet-like cell clusters (ICCs) and sevenfold lower in purified beta-cells compared with adult islets in the pig. The low level of enzyme activity was a result of low gene expression of the mitochondrial enzymes in the fetal beta-cells. Increasing NADH shuttle activity by transduction of fetal rat islets with mGPDH cDNA enabled the fetal islets to secrete insulin when stimulated with glucose. We concluded that the immaturity of the NADH shuttles contributes to the inability of fetal beta-cells to secrete insulin in response to glucose.

Effect of several anions on the activity of mitochondrial malate dehydrogenase from pig heart

Mitochondrial malate dehydrogenase (mMDH) shows a complex dependence upon ionic environment that includes kinetic and structural effects. We measured mMDH activity in several buffers (phosphate, MOPS, and MES) at pH 6.5 and 7.5, and in the presence of a number of anions, at highly diluted enzyme concentrations where mMDH showed significant loss of activity. Under these conditions, mMDH activity shows a non-linear dependence on enzyme concentration, in agreement with the existence of a dimer–monomer equilibrium, where only the dimeric form is active. According to this hypothesis, the dissociation constant of mMDH dimer has been determined to be 5.4 nM in the MES buffer at pH 6.5. Either the presence of a small anion like phosphate, or an increase of the pH from 6.5 to 7.5 shifts the equilibrium in favor of the dimeric form with the two effects appearing to be additive. To extend the study, we analysed the effect of a number of anions on the mMDH activity in 50 mM MOPS buffer at pH 7.5. All the anions had a dual effect: at low concentrations, they increased the activity of mMDH, while at high concentrations, they inhibited it. A more accurate analysis of the data revealed that the activation capacity of all the anions tested was similar, although they differed in their inhibitory influence. To show these differences more clearly, the experiment was repeated in 50 mM phosphate buffer at pH 7.5, under conditions where almost all activations were due to the buffer. The analysis of the results obtained under these conditions revealed the following sequence of inhibition potency: phosphate<acetate<sulfate<chloride<bromide<nitrate<perchlorate<thiocyanate, that was in good agreement with their chaotropicity. The behaviour of mMDH in the presence of different ions and/or ionic strength indicates that at low ionic concentrations, increased activity is likely due to a stabilization of the dimeric form of the enzyme. Further increases in the ionic concentration, especially if chaotropic ions are used, lead to a loss of enzyme activity that appears to be a consequence of structural changes in the enzyme rather than kinetic effects.

Kinetics of thyroid hormone induced changes in liver and skeletal muscle enzymes of Clarias batrachus.

Kinetics of triiodothyronine (T3) induced changes were studied in cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) of the liver and skeletal muscle of a catfish, Clarias batrachus. The rates of gradual inductions in the activities of all the three metabolic enzymes were faster in skeletal muscle than those of the liver. These time-dependent and tissue-specific inductions may be due to the possible differences in the rates of different enzymic syntheses. The maximum inductions in the activities of cMDH, mMDH and LDH were recorded around 19 hr after T3 treatment. Thereafter, the activities of all the enzymes gradually declined to their half levels within the next 12 hr which reflected the physiological half-life of these metabolic enzymes in the freshwater catfish.

Impact of Endosulfan on Cytoplasmic and Mitochondrial Liver Malate Dehydrogenase from the Freshwater Catfish ( Clarias batrachus)

The impact of a sublethal concentration of an organochlorine pesticide endosulfan on the activity, specific activity, electrophoretic patterns and kinetic properties of crude and purified forms of cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH) was evaluated in the liver of the freshwater catfish, Clarias batrachus. The endosulfan reduced significantly the activity and the specific activity of cMDH and mMDH, but had no effect on total cytoplasmic and mitochondrial protein contents. The inhibition produced by endosulfan was of mixed non-competitive-uncompetitive type (K iE > K iES) and of mixed competitive-non-competitive type (K iE < K iES) for crude cMDH and mMDH, respectively. The PAGE shows five distinct isoforms (C1 to C5) of cMDH and two isoforms (M1 and M2) of mMDH. The C5-isoform of liver cMDH is predominant and it corresponds to M2-isoform of mMDH. There are no endosulfan-associated differences in the relative charges of crude cMDH and mMDH as well as their purified isoforms, C5-cMDH and M2-mMDH. The relative molecular weights of the purified isoforms are not affected by endosulfan. The purified C5-cMDH and M2-mMDH of endosulfan-treated liver in vivo showed simple non-competitive (K iE = K iES) type of inhibition; whereas in vitro it was of uncompetitive (K iES) and mixed competitive-non-competitive (K i < K iES) type for the two respective isoforms. G-1-P acts as an uncompetitive (K iES) inhibitor of C5-cMDH and mixed competitive-non-competitive (K iE < K iES) inhibitor of M2-mMDH of the control fish. The inhibitory pattern of G-1-P is modulated by endosulfan in case of C5-cMDH; whereas there is no alteration in case of M2-mMDH. Summarizing, it can be stated that endosulfan exerts an inhibitory effect on crude cMDH and mMDH in vivo, and their purified isoforms (C5-cMDH and M2-mMDH) in vivo as well as in vitro. The impact of endosulfan is mediated through enzyme-substrate-endosulfan (ES-END) complexing for cMDH and enzyme-endosulfan (E-END) complexing for mMDH.

Intra-acinar profiles of cytosolic and mitochondrial malate dehydrogenase isoenzymes in rat liver.

A new method for electrophoretic separation of the isoforms of malate dehydrogenase in microdissected tissue samples was applied to rat liver. The intra-acinar activity profiles of cytosolic (cMDH) and mitochondrial (mMDH) malate dehydrogenase were determined in male and female control animals and in animals fasted for 84 hr. Measurements were carried out on lyophilized liver tissue samples of 50-100 ng from the whole length of the sinusoid. The results showed that both in fed and fasted animals, mMDH activity was almost evenly distributed throughout the acinus in livers of both sexes. cMDH showed higher activity in the periportal area compared with the perivenous area by a factor of approximately 1.35 in all animals studied. Our results favor a slightly higher capacity of the malate-aspartate shuttle in the periportal area in both fed and fasted animals. Furthermore, the distribution pattern of mMDH suggests that this isoenzyme is not a marker for the zonation of the oxidative metabolism in the liver acinus.

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.

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME.