The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

Purification and biochemical properties of genetically defined malate dehydrogenase in maize

Malate dehydrogenase of maize exists in multiple molecular forms (isozymes). In strain W64A, two soluble forms (s-MDH), five mitochondrial forms (m-MDH), and two glyoxysomal forms (g-MDH) were found in etiolated seedlings. The s-MDHs and m-MDHs were prepared in highly purified form. Using these purified isozymes, experiments with reducing agents (100 mm mercaptoethanol), low pH (2.0), and high salt cocn (7.5 m guanidine-HCl), along with genetic data, have eliminated the possibility of conformational alterations as an explanation for MDH multiplicity in maize; the MDH isozymes are genetically determined. Biochemical properties for each of the seven MDH isozymes were examined. Molecular weight, pI, pH optimum, thermolability, and K_m for oxaloacetate, malate, NAD, and NADH at different pH values were determined for each isozyme. Different kinetics of substrate inhibition (oxaloacetate) and coenzyme inhibition (NAD) were observed for the different isozymes. Effects of NAD analogs, chelating agents, reducing agents, metal ions, and TCA cycle acids on the enzymatic activity of these isozymes were tested. Based on the physical and kinetic properties observed, the maize malate dehydrogenase isozymes can be classified into four groups: s-MDH¹; s-MDH²; the two most anodal m-MDHs; and the three most cathodal m-MDHs. Since strain W64A is highly inbred, our data along with our previous and simultaneous genetic analysis suggest that multiple genes are involved in the expression of maize malate dehydrogenase isozymes.     

Identification of cytoplasmic nodule-associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum

The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion-exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak. During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity. The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The Km values for malate (2.6 mM), NAD+ (27 microM), oxaloacetate (18 microM) and NADH (13 microM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 microM and 70 microM respectively). Binding of malate by the enzyme-NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex. From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacteroid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration. Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.     

Plasmalemma-associated malate dehydrogenase activity in onion root cells

Summary Plasma membrane vesicles isolated from onion roots showed oxaloacetate reductase activity as well as other oxidoreductase activities. Purification and further sequencing showed that the protein responsible for the activity is a 40 kDa protein which corresponds to the cytosolic soluble malate dehydrogenase. However, the activity remained bound to the membrane after repeated freezing and thawing cycles and further washing, excluding a cytosolic contamination as the source of the activity. Furthermore, a second 28 kDa protein has been copurified together with the 40 kDa protein. The plasmalemma oxaloacetate reductase activity shows both donor and acceptor sites located towards the cytoplasmic side of the plasma membrane. This enzyme catalyzed the oxidation of NADH by oxaloacetate and the reduction of NAD⁺ by malate in the presence of an oxaloacetate-withdrawing system. We conclude that a significant amount of the cytosolic malate dehydrogenase can be specifically attached to the cytosolic face of the plasmalemma. A possible role in a putative malate shuttle associated to the plasma membrane is discussed.Abbreviations AFR ascorbate free radical - DQ duroquinone - OA oxaloacetate - DPIP dichlorophenolindophenol - MDH malate dehydrogenase - PHMB p-hydroxymercuribenzoate     

Fluorometric determination of alpha-ketosuccinamic acid in rat tissues

A method for the fluorometric determination of alpha-ketosuccinamic acid, the alpha-keto acid analog of asparagine, is described. The procedure involves the hydrolysis of alpha-ketosuccinamate to oxaloacetate by omega-amidase followed by NADH-dependent reduction of oxaloacetate to malate by malate dehydrogenase. A correction for endogenous oxaloacetate is made by using control samples lacking omega-amidase. Of the rat tissues investigated, liver contained the highest concentration, followed by kidney (53 +/- 6 (n = 11) and 18 +/- 3 (n = 3) mumol/kg wet wt, respectively). alpha-Ketosuccinamate was not detected in brain (less than 8 mumol/kg wet wt). Some chemical properties of alpha-ketosuccinamate were investigated. Concentrated solutions of sodium alpha-ketosuccinamate frozen for extended periods and the solid sodium salt of alpha-ketosuccinamate dimer heated to 130 degrees C are converted to at least 10 products by processes involving dimerization, dehydration, and decarboxylation. Isobutane chemical ionization mass spectral analysis (170-230 degrees C) of the free acid monomer yielded similar products. Many of the breakdown products were identified as di- and monoheterocyclic compounds, some of which are known to be of biological importance.     

Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians

Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica.     

Electrophoretic patterns and thermostability of some proteins from heat-hardened wheat

Heat hardening of wheat (Triticum aesticum L.) resulted in increased heat stability in leaf extracts of fraction I protein, malate dehydrogenase and peroxidase. Polyacrylamide disk gel electrophoretic atterns of fraction 1 protein, malate dehydrogenase and peroxidase did not change after the heat hardening. The individual isozymes of malate dehydrogenase and peroxidase were found to possess the different heat stabilities. After heat hardening only some of the isozymes had an increased heat stability.     

Functional interrelations between isozymes of dehydrogenases in the intact and denervated rabbit muscles]

A correlation is shown to exist between malate dehydrogenase (MDH), lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (glycerol-3-PDH activity values, lactate/pyruvate and malate/oxaloacetate coefficients, MDH and LDH isozyme spectra and kinetic properties of LDH isozymes in soluble fractions of cytoplasm from intact rabbit m. soleus (red), m. gastrocnemius (mixed) and m. quadratus lumborum (white). In denervated soleus and gastrocnemius the cytoplasmic MDH/LDH, mitochondrial MDH/LDH, MDH mitochondrial/MDH cytoplasmic activity ratios, concentrations of substrates and isozyme spectra of MDH and LDH tend to equalize. The obtained results indicate the importance of isozyme composition and total activity ratios of the dehydrogenases for regulation of pyruvate and NADH metabolic pathways.     

Malate dehydrogenase isozymes in the longnose dace,Rhinichthys cataractae

The interspecies homology of dace supernatant (A₂, AB, B₂) and mitochondrial (C₂) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.This research was supported in part by NSF Grant SM176-83974 and a grant from the Blakeslee Fund.     

Isozymes in wheat-barley hybrid derivative lines

Zymogram analysis was used to identify the barley chromosomes that carry the structural genes for particular isozymes. Wheat, barley, and wheatbarley hybrid derivative lines (which contained identified barley chromosomes) were tested by gel electrophoresis for isozymes of particular enzymes. It was found that barley chromosome 4 carries structural genes for acid phosphatase and amylase isozymes, barley chromosome 5 carries genes for phosphoglucose isomerase and malate dehydrogenase isozymes, and that barley chromosome 2 carries a gene for at least one glucose-6-phosphate dehydrogenase protomer. These results reinforce previous conclusions that barley chromosome 4 shows homoeology with wheat chromosome group 4 and that barley chromosome 5 shows homoeology with wheat chromosome group 1.     

Regulation of malate oxidation in isolated mung bean mitochondria: I. Effects of oxaloacetate, pyruvate, and thiamine pyrophosphate

In order to investigate the relationship between malate oxidation and subsequent cycle reactions, the effects of oxaloacetate, pyruvate, and thiamine pyrophosphate on malate oxidation in mung bean (Phaseolus aureus var. Jumbo) hypocotyl mitochondria were quantitatively examined. Malate oxidation was optimally stimulated by addition of pyruvate and thiamine pyrophosphate, whose addition lowered the apparent Km for malate from 5 mm to 0.1 mm. Intermediate analysis showed that the stimulatory effect was correlated with removal of oxaloacetate to citrate. Oxaloacetate added alone was shown not to be metabolized until addition of pyruvate and thiamine pyrophosphate; then oxaloacetate was converted in part to pyruvate and also to citrate. These results establish that malate oxidation in mung bean mitochondria is subject to control by oxaloacetate levels, which are primarily determined by the resultant of the activities of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase.     

The control of wheat malate dehydrogenase by rye chromosomes

A quantitative analysis of malate dehydrogenase isozymes has been carried out in a hexaploid wheat Triticum aestivum variety Holdfast, a diploid rye Secale cereale variety King II, a series of seven addition lines each having the Holdfast wheat chromosome complement, and also a different homologous pair of King II rye chromosomes. In young shoots of three of these addition lines grown in a defined salts medium lacking sucrose, at least one isozyme activity was elevated. This did not occur in shoots grown in a medium containing 0.5% sucrose or in the Triticale possessing the full wheat and rye chromosomal complements grown in the absence of exogenous sucrose. On the basis of cellular localization and substrate inhibition studies, the particular isozyme activities enhanced by the rye chromosomes were indistinguishable from isozyme activities in Holdfast wheat and dissimilar to all malate dehydrogenase isozyme activities observed in King II rye. These results suggest that three different rye chromosomes produce gene products which can interact with the wheat malate dehydrogenase regulatory system.     

Kinetic behaviour of chicken liver mitochondrial malate dehydrogenase and lactate dehydrogenase mixtures

• 1.1. In the mitochondria of chicken liver cells there is lactate dehydrogenase activity that catalyses the reduction of the oxaloacetate by the NADH.
• 2.2. The presence of lactate dehydrogenase in the malate dehydrogenase preparations causes an apparent activation in the double-reciprocal plot at high oxaloacetate concentrations that depends on the lactate dehydrogenase/malate dehydrogenase ratio in the preparation.
• 3.3. The separation of the two molecular forms of chicken liver mitochondrial malate dehydrogenase, free from lactate dehydrogenase, is described.

     

A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V_max with no apparent effect on K_m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.     

Thermal kinetic differences between allelic isozymes of malate dehydrogenase (Mdh-B locus) of largemouth bass,Micropterus salmoides

Two alleles are encoded at the malate dehydrogenase locus in largemouth bass, Micropterus salmoides. Populations in the extreme northern areas of the range of this fish are fixed or nearly fixed for the B1 allele, whereas populations in Florida are fixed for the alternative allele, B2. The MDH-B1B1 and MDH-B2B2 allelic isozymes were isolated by preparative starch gel electrophoresis and subjected to in vitro kinetic analyses. The apparent Km (oxaloacetate) for each of these allelic isozymes was determined at 25, 30, and 35 degrees C. The Km values for both isozymes increased with increasing temperature and were not significantly different from each other at 25 and 35 degrees C. However, at 30 degrees C the Km value for the MDH-B1B1 allelic isozyme was higher than that for the MDH-B2B2 isozyme (i.e., 5.4 X 10(-5) vs 3.3 X 10(-5)). These results are consistent with the hypothesis that the different environmental temperatures at different latitudes may be at least partially responsible for the north-south cline in Mdh-B allele frequencies.     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn²⁺-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.     

The control of malate dehydrogenase activity by adenine nucleotides in purified potato tuber (Solanum tuberosum L.) mitochondria

The limiting factors of the involvement of malate dehydrogenase in mitochondrial malate oxidation were investigated by using Percoll-purified potato tuber mitochondria. The respective roles of reduced pyridine nucleotides, oxaloacetate, and adenine nucleotides were studied under conditions of high or low phosphorylation potential (Pi + ADP/ATP ratio). Under conditions of high phosphorylation potential, the limitation of malate dehydrogenase activity was caused by the accumulation of oxaloacetate in the medium. In the absence of ADP (phosphorylation potential close to zero), ATP was responsible for the inhibition of malate dehydrogenase activity rather than oxaloacetate or reduced pyridine nucleotides.     

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.     

Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase.

The mechanistic implications of the kinetic behaviour of a fusion protein of mitochondrial malate dehydrogenase and citrate synthase have been reanalysed in view of predictions based on experimentally determined kinetic parameter values for the dehydrogenase and synthase activities of the protein. The results show that the time-course of citrate formation from malate in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is taken to the inhibitory effects of NADH and oxaloacetate on the malate dehydrogenase activity. The effect of aspartate aminotransferase on the coupled reaction is likewise fully consistent with that expected for a free-diffusion mechanism. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses citrate formation from malate by a mechanism involving channelling of the intermediate oxaloacetate.