Regulation of malate dehydrogenase activity by glutamate, citrate, alpha-ketoglutarate, and multienzyme interaction

Binding experiments indicate that mitochondrial aspartate aminotransferase can associate with the alpha-ketoglutarate dehydrogenase complex and that mitochondrial malate dehydrogenase can associate with this binary complex to form a ternary complex. Formation of this ternary complex enables low levels of the alpha-ketoglutarate dehydrogenase complex, in the presence of the aminotransferase, to reverse inhibition of malate oxidation by glutamate. Thus, glutamate can react with the aminotransferase in this complex without glutamate inhibiting production of oxalacetate by the malate dehydrogenase in the complex. The conversion of glutamate to alpha-ketoglutarate could also be facilitated because in the trienzyme complex, oxalacetate might be directly transferred from malate dehydrogenase to the aminotransferase. In addition, association of malate dehydrogenase with these other two enzymes enhances malate dehydrogenase activity due to a marked decrease in the Km of malate. The potential ability of the aminotransferase to transfer directly alpha-ketoglutarate to the alpha-ketoglutarate dehydrogenase complex in this multienzyme system plus the ability of succinyl-CoA, a product of this transfer, to inhibit citrate synthase could play a role in preventing alpha-ketoglutarate and citrate from accumulating in high levels. This would maintain the catalytic activity of the multienzyme system because alpha-ketoglutarate and citrate allosterically inhibit malate dehydrogenase and dissociate this enzyme from the multienzyme system. In addition, citrate also competitively inhibits fumarase. Consequently, when the levels of alpha-ketoglutarate and citrate are high and the multienzyme system is not required to convert glutamate to alpha-ketoglutarate, it is inactive. However, control by citrate would be expected to be absent in rapidly dividing tumors which characteristically have low mitochondrial levels of citrate.     

Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase

Current evidence suggests that mitochondrial matrix enzymes exist in solid-state, multienzyme complexes in vivo. Addition of polyethylene glycol to a solution containing malate dehydrogenase and citrate synthase generates such a solid-state, enzyme complex in vitro at enzyme concentrations permitting kinetic measurements. Suspensions of the isolated, solid-state, hetero-complex of these enzymes were used to study the coupled reactions of citrate synthesis from malate, NAD, and CoASAc. The particles appear to be about 1 microgram in diameter. Considering the ratio of enzyme to oxalacetate molecules in or at the surface of the solid-state particles, one would expect oxalacetate to be converted to citrate within a few molecular distances of the site of oxalacetate generation. This model of "substrate channeling" (or alternatively a direct transfer of oxalacetate between enzymes) is supported by experiments with excess aspartate aminotransferase and glutamate added to the solution phase to give a reaction competing with the synthase for bulk phase oxalacetate. Quantities of aminotransferase that reduce the citrate reaction rate with soluble dehydrogenase and synthase by 90% do not significantly affect rates with comparable amounts of the dehydrogenase-synthase complex. We suggest that similar substrate channeling can occur in vivo and discuss the possible advantages provided thereby.     

Interaction between citrate synthase and mitochondrial malate dehydrogenase in the presence of polyethylene glycol

Pig heart citrate synthase and mitochondrial malate dehydrogenase interact in polyethylene glycol solutions as indicated by increased solution turbidity. A large percentage of both enzymes sediments when mixtures of the two in polyethylene glycol are centrifuged, whereas little if any of either enzyme sediments in the absence of the other. The observed interaction is highly specific in that neither cytosolic malate dehydrogenase nor nine other proteins showed evidence of specific interaction with either pig heart citrate synthase or mitochondrial malate dehydrogenase. Escherichia coli citrate synthase did not interact with pig heart citrate synthase, but did show evidence of interaction with pig heart mitochondrial malate dehydrogenase. The relation between enzyme behavior in polyethylene glycol solution and in the mitochondrion and the significance of possible in vivo interactions between citrate synthase and mitochondrial malate dehydrogenase are discussed.     

Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes.

The level of aspartate aminotransferase in liver mitochondria was found to be approximately 140 microM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (M(r) = 1.6 x 10(5) to 2.7 x 10(6)) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase-binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes. The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase. The level of malate dehydrogenase was found to be so high (140 microM) in liver mitochondria, compared with that of citrate synthase (25 microM) and the pyruvate dehydrogenase complex (0.3 microM), that there would also be a sufficient supply of oxalacetate to citrate synthase-pyruvate dehydrogenase.     

Interactions between carbamyl phosphate synthase-I-mitochondrial aspartate aminotransferase and palmitoyl-CoA

Carbamyl phosphate synthase-I and glutamate dehydrogenase both form a complex with mitochondrial aspartate aminotransferase. Instead of these two enzymes competing for the aminotransferase, carbamyl phosphate synthase-I enhances glutamate dehydrogenase-aminotransferase interaction. This suggests that a complex can be formed between all three enzymes. Since this complex is stable in the presence of substrates and modifiers of the three enzymes, it could conceivably convert NH₄⁺ produced from aspartate into carbamyl phosphate. Furthermore, since carbamyl phosphate synthase-I is the predominant protein in liver mitochondria, it could play a major role in placing the aminotransferase and glutamate dehydrogenase in close proximity. Malate removes glutamate dehydrogenase from the tri-enzyme complex and thus could play a role in determining whether glutamate dehydrogenase interacts with carbamyl phosphate synthase-I or is available to participate in reactions with the Krebs cycle. Palmitoyl-CoA has a high affinity for both carbamyl phosphate synthase-I and glutamate dehydrogenase. ATP and malate which, respectively, decrease and enhance binding of palmitoyl-CoA to glutamate dehydrogenase, respectively decrease and enhance the ability of this enzyme to compete with carbamyl phosphate synthase-I for palmitoyl-CoA. Since carbamyl phosphate synthase-I is present in high levels in liver mitochondria and has a high affinity for palmitoyl-CoA, it could play a major role as a reservoir for palmitoyl-CoA.     

Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

Glutamate dehydrogenase--malate dehydrogenase complex.

Kinetic and Sephadex gel filtration epxeriments indicate that in the presence of palmitoyl-CoA, glutamate dehydrogenase forms a complex with mitochondrial malate dehydrogenase. In this complex, palmitoyl-CoA is bound to glutamate dehydrogenase but is not bound to malate dehydrogenase. Consequently, palmitoyl-CoA inhibits glutamate dehydrogenase while glutamate dehydrogenase completely protects malate dehydrogenase activity against palmitoyl-CoA inhibition. In the absence of palmitoyl-CoA, interaction between these two enzymes is quite weak. However, if the two enzymes are incubated with the bifunctional crosslinker dimethyl 3,3′-dithiobispropionimidate and chromatographed on Sephadex G-200, about 46% of the malate dehydrogenase is eluted with glutamate dehydrogenase in the void volume. If glutamate dehydrogenase or crosslinker is omitted, then malate dehydrogenase is not found in the void volume or other early fractions from the column. This indicates that in the absence of palmitoyl-CoA the crosslinker prevents dissociation of the weak complex by forming a covalent bond between the two enzymes. Furthermore, if the two enzymes are incubated in polyethylene glycol, there is a marked increase in the amount of both enzymes precipitated.     

Polyethylene glycol-induced heteroassociation of malate dehydrogenase and citrate synthase

Studies by dynamic and total intensity light scattering, ultracentrifugation, electron microscopy, and chemical crosslinking on solutions of the pig heart mitochondrial enzymes, malate dehydrogenase and citrate synthase (separately and together) demonstrate that polyethylene glycol induces very large homoassociations of each enzyme, and still larger heteroenzyme complexes between these two enzymes in the solution phase. Specificity of this heteroassociation is indicated by the facts that heteroassociations with bovine serum albumin were not observed for either the mitochondrial dehydrogenase or the synthase or between cytosolic malate dehydrogenase and citrate synthase. The weight fraction of the enzymes in the mitochondrial dehydrogenase-synthase associated particles in the solution phase was less than 0.03% with the dilute conditions used in the dynamic light scattering measurements. Neither palmitoyl-CoA nor other solution conditions tested significantly increased this weight fraction of associated enzymes in the solution phase. Because of the extremely low solubility of the associated species, however, the majority of the enzymes can be precipitated as the heteroenzyme complex. This precipitation is a classical first-order transition in spite of the large particle sizes and broad size distribution. Ionic effects on the solubility of the heteroenzyme complex appear to be of general electrostatic nature. Polyethylene glycol was found to be more potent in precipitating this complex than dextrans, polyvinylpyrrolidones, ficoll, and beta-lactoglobulin.     

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.     

Regulation of aminotransferase-glutamate dehydrogenase interactions by carbamyl phosphate synthase-I, Mg2+ plus leucine versus citrate and malate

Citrate, malate, and high levels of ATP dissociate the mitochondrial aspartate aminotransferase-glutamate dehydrogenase complex and have an inhibitory effect on the latter enzyme. These effects are opposed by Mg2+, leucine, Mg2+ plus ATP, and carbamyl phosphate synthase-I. In addition, Mg2+ directly facilitates formation of a complex between glutamate dehydrogenase and the aminotransferase and displaces the aminotransferase from the inner mitochondrial membrane which could enable it to interact with glutamate dehydrogenase in the matrix. Zn2+ also favors an aminotransferase-glutamate dehydrogenase complex. It, however, is a potent inhibitor of and has a high affinity for glutamate dehydrogenase. Leucine, however, enhances binding of Mg2+ and decreases binding of and the effect of Zn2+ on the enzyme. Thus, since both metal ions enhance enzyme-enzyme interaction and Zn2+ is a more potent inhibitor, the addition of leucine in the presence of both metal ions results in activation of glutamate dehydrogenase without disruption of the enzyme-enzyme complex. Furthermore, the combination of leucine plus Mg2+ produces slightly more activation than leucine alone. These results indicate that leucine, carbamyl phosphate synthase-I, and its substrate and cofactor, ATP and Mg2+, operate synergistically to facilitate glutamate dehydrogenase activity and interaction between this enzyme and the aminotransferase. Alternatively, Krebs cycle intermediates, such as citrate and malate, have opposing effects.     

Inhibition of glutamate dehydrogenase and malate dehydrogenases by palmitoyl coenzyme A.

In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA.     

Metabolic zonation in teleost gastrointestinal tract

Activities of several metabolic enzymes show distinct patterns of zonation along the intestinal tract of tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and copper rockfish (Sebastes caurinus). Zonation is species and enzyme specific, with different metabolic activities concentrated in specific areas, and few generalizations can be made. The rockfish show the smallest degree of zonation, with highest activities in the third quarter of the intestine, and shallow gradients to either side, and a general upswing in activity towards the distal end. In the trout, mitochondrial enzyme activities (citrate synthase, glutamate dehydrogenase, malate dehydrogenase) are highest in the pyloric caeca and decrease along the length of the small intestine. This pattern is accentuated for malic enzyme and glucose 6-phosphate dehydrogenase. These enzymes drop precipitously in activity after the first few sections of the small intestine, while other NADP-linked dehydrogenases (isocitrate dehydrogenase, and 6-phosphogluconate dehydrogenase) show moderate activity in pyloric caeca and peak toward the distal section of the small intestine. In tilapia, glutamate dehydrogenase shows a similar decrease as in trout, but citrate synthase peaks towards the distal sections. NADP-dependent dehydrogenases reveal distinct patterns, peaking in different sections of the intestine-malic enzyme in the proximal midsection, glucose 6-phosphate dehydrogenase in the distal mid-section, and isocitrate dehydrogenase in the anal section. Enzyme activities in the stomach of trout and tilapia also show zonation, with the midsection generally displaying the highest activities. A 5-day treatment of tilapia with an intraperitoneal cortisol deposit (25 mg kg(-1) wet mass) drastically alters metabolic performance along the gut in enzyme specific patterns, generally increasing enzyme activities in site-specific arrangements. Cortisol treatment also leads to the expected increases in activities of phosphoenolpyruvate carboxykinase, pyruvate kinase and aspartate aminotransferase in liver, but not in kidney. Aspartate aminotransferase is the only enzyme in brain significantly increased by cortisol treatment. Short-term food deprivation changes enzyme patterns, often resembling those observed after cortisol administration. We conclude that brain, liver and intestinal amino acid metabolism is an important target for cortisol action in fish and that metabolic zonation is a key factor to be reckoned with when analyzing physiological phenomena in the fish intestine.     

Regulation of glutamate dehydrogenase by palmitoyl-coenzyme A

Glutamate dehydrogenase is inhibited more by palmitoyl-CoA when the reduced form of triphosphopyridine nucleotide instead of the reduced form of diphosphopyridine nucleotide is the coenzyme. Inhibition is further enhanced by α-ketoglutarate and malate. Thus, for example, in the presence of TPNH plus malate, the amount of palmitoyl-CoA required for 50% inhibition is 10-fold lower (0.03 μm) than previously reported values obtained with reduced diphosphopyridine nucleotide as a coenzyme. Allosteric modifiers such as ATP, GTP, and leucine decrease inhibition of glutamate dehydrogenase by palmitoyl-CoA. Palmitoyl-CoA and ADP are competitive. Thus, the palmitoyl-CoA binding site is apparently in the vicinity of the site of these allosteric modifiers and is probably at the ADP site. The fact that ADP (which has only one site per polypeptide chain) can completely prevent inhibition by palmitoyl-CoA suggests that there is only one kinetically significant palmitoyl-CoA binding site per polypeptide chain. This is consistent with the fact that adding one equivalent of palmitoyl-CoA per polypeptide chain inhibits about 80%. The high affinity of glutamate dehydrogenase for palmitoyl-CoA enables it to successfully compete with other mitochondrial proteins for palmitoyl-CoA.     

Effect of aspartate on complexes between glutamate dehydrogenase and various aminotransferases.

In previous studies it was found that: (a) aspartate aminotransferase increases the aspartate dehydrogenase activity of glutamate dehydrogenase; (b) the pyridoxamine-P form of this aminotransferase can form an enzyme-enzyme complex with glutamate dehydrogenase; and (c) the pyridoxamine-P form can be dehydrogenated to the pyridoxal-P form by glutamate dehydrogenase. It was therefore concluded (Fahien, L.A., and Smith, S.E. (1974) J. Biol. Chem 249, 2696-2703) that in the aspartate dehydrogenase reaction, aspartate converts the aminotransferase into the pyridoxamine-P form which is then dehydrogenated by glutamate dehydrogenase. The present results support this mechanism and essentially exclude the possibility that aspartate actually reacts with glutamate dehydrogenase and the aminotransferase is an allosteric activator. Indeed, it was found that aspartate is actually an activator of the reaction between glutamate dehydrogenase and the pyridoxamine-P form of the aminotransferase. Aspartate also markedly activated the alanine dehydrogenase reaction catalyzed by glutamate dehydrogenase plus alanine aminotransferase and the ornithine dehydrogenase reaction catalyzed by ornithine aminotransferase plus glutamate dehydrogenase. In these latter two reactions, there is no significant conversion of aspartate to oxalecetate and other compounds tested (including oxalacetate) would not substitute for aspartate. Thus aspartate is apparently bound to glutamate dehydrogenase and this increases the reactivity of this enzyme with the pyridoxamine-P form of aminotransferases. This could be of physiological importance because aspartate enables the aspartate and ornithine dehydrogenase reactions to be catalyzed almost as rapidly by complexes between glutamate dehydrogenase and the appropriate mitochondrial aminotransferase in the absence of alpha-ketoglutarate as they are in the presence of this substrate. Furthermore, in the presence of aspartate, alpha-ketoglutarate can have little or no affect on these reactions. Consequently, in the mitochondria of some organs these reactions could be catalyzed exclusively by enzyme-enzyme complexes even in the presence of alpha-ketoglutarate. Rat liver glutamate dehydrogenase is essentially as active as thebovine liver enzyme with aminotransferases. Since the rat liver enzyme does not polymerize, this unambiguously demonstrates that monomeric forms of glutamate dehydrogenase can react with aminotransferases.     

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.     

Physiological differentiation of the mitochondria during Bufo bufo development.

1) The oxygen consumption increases during Bufo bufo development in accordance with the two steps which border at the "heart beat" stage. 2) Cytochrome c oxidase activity is not proportional to the oxygen consumption: it is notable and constant in the first step, and it only increases in the second. 3) In the mitochondria of preneural embryos, citrate synthase, NADP+ dependent isocitrate dehydrogenase, and succinate dehydrogenase activities are very low in respect to malate dehydrogenase and glutamate oxaloacetate transaminase activities. The Krebs cycle results lowered at the condensing reaction level with acetyl accumulation when pyruvate is available. The same behavior has been observed in the Xenopus laevis oocytes and differentiated tissues. 4) The presence of a phosphagen system which is different from creatine phosphate and arginine phosphate, supporting ATP level, has been demonstrated in B. bufo embryos. 5) Mitochondria of postneural embryos are able to accomplish a complete Krebs cycle by increasing citrate synthase, and succinate dehydrogenase activities. 6) In all B. bufo development, malate dehydrogenase and glutamate oxaloacetate transaminase constitute a multienzymatic system by which the mitochondria accomplish a decarboxylic amino acid shunt required for the transformation of deutoplasm into protoplasm. This shunt is also operative in the X. laevis oocytes. 7) Through pyruvate production, by oxidative decarboxylation of malate, the NAD(P)+ dependent malic enzyme could carry out a fundamental anaplerotic function in the mitochondria which is specialized in the production of biosynthetic blocks belonging to the embryo in which the carbohydrates metabolism rather than the glycolytic activity is designed for pentose phosphate and glycerol phosphate synthesis for protein and cytomembrane production. 8) Consistent metabolic differences have been highlighted between B. bufo embryos and X. laevis embryos.     

Physiology and metabolism of two alkane oxidizing and citric acid producing strains of Candida parapsilosis

Summary The activity of enzymes of the tricarboxylic acid (TAC) and glyoxylate (GC) cycles in Candida parapsilosis (wild type KSh 21 and mutant 337) were studied under different physiological and metabolic conditions. C. parapsilosis differed in most of its enzyme activities from other non-citric acid producing yeasts. Furthermore, pH-value, temperature and age of culture proved to act differently on both strains of the tested organism.The addition of trans-aconitate increased not only the growth but also the activities of citrate synthase and some other enzymes while that of aconitase decreased enormously.The high citrate synthase activity might be connected with the role of citrate in the transport of acetyl groups.Abbreviations CS citrate synthase - AC aconitase - ICDH isocitrate dehydrogenase - GDH glutamate dehydrogenase - Fum fumarase - MDH malate dehydrogenase - ICL isocitrate lyase - MS malate synthase     

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.     

Thermodynamic resolution of cytochrome P450 and characterization of an iron-sulfur center in rat liver microsomal preparations.

Binding of 8-anilinonaphthalene sulfonate (ANS) to glutamate dehydrogenase results in enzyme inhibition and a marked increase in the fluorescence of ANS. Perphenazine and GTP increase the fluorescence of ANS-glutamate dehydrogenase secondary to their known ability to alter the conformation of this enzyme. Aspartate aminotransferases, which form enzyme-enzyme complexes with glutamate dehydrogenase, produce a slight decrease in the fluorescence of ANS-glutamate dehydrogenase.While ANS and perphenazine are allosteric inhibitors of reactions catalyzed by free glutamate dehydrogenase, they do not inhibit reactions catalyzed by aminotransferaseglutamate dehydrogenase complexes. This is in spite of the fact that the aminotransferase does not prevent either ANS or perphenazine from being bound to glutamate dehydrogenase. Therefore, reactions catalyzed by the enzyme-enzyme complex are apparently not inhibited by ANS or perphenazine because binding of the aminotransferase to glutamate dehydrogenase prevents these ligands from altering the conformation of glutamate dehydrogenase. This is consistent with the fact that the aminotransferase also prevents perphenazine from enhancing the fluorescence of ANS-glutamate dehydrogenase.Reactions catalyzed by the enzyme-enzyme complex are inhibited by GTP and the aminotransferase does not prevent GTP from enhancing the fluorescence of ANS-glutamate dehydrogenase. Therefore, binding of the aminotransferase to glutamate dehydrogenase does not prevent GTP from altering the conformation of glutamate dehydrogenase.The fact that the aminotransferase completely prevents perphenazine from increasing the fluorescence of ANS-glutamate dehydrogenase suggests that in the enzymeenzyme complex each glutamate dehydrogenase polypeptide chain can be bound to an aminotransferase polypeptide chain. This would mean that three aminotransferase molecules can be bound to each monomeric unit (M_r 3 × 10⁵) of glutamate dehydrogenase.     

Regulation of mitochondrial malate dehydrogenase: kinetic modulation independent of subunit interaction

Porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37), a dimeric enzyme of Mr = 70,000, is both allosterically activated and inhibited by citrate. Using an affinity elution procedure based upon citrate binding to malate dehydrogenase, the isolation of pure heterodimer (a dimeric species with one active subunit and one iodoacetamide-inactivated subunit) has been achieved. Investigations utilizing this heterodimer in conjunction with resin-bound monomers of malate dehydrogenase have allowed the formulation of a definite conclusion concerning the role of subunit interactions in catalysis and regulation of this enzyme. The citrate kinetic effects, oxaloacetate inhibition, malate activation, and the effects of 2-thenoyl-trifluoroacetone (TTFA) are shown to be independent of interaction between catalytically active subunits. Previous kinetic data thought to support a reciprocating catalytic mechanism for this enzyme may be reinterpreted upon closer analysis in relation to an allosteric, conformationally specific binding model for malate dehydrogenase.