Pig Heart Mitochondrial Malate Dehydrogenase Research Articles

Interaction between Citrate Synthase and Malate Dehydrogenase: SUBSTRATE CHANNELING OF OXALOACETATE

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase.

Thermodynamic analysis of the stabilisation of pig heart mitochondrial malate dehydrogenase and maize leaf phospho enolpyruvate carboxylase by different salts, amino acids and polyols

As part of our investigations into the inactivation of pig heart mitochondrial malate dehydrogenase ( phm-MDH) and maize leaf phospho enolpyruvate carboxylase ( ml-PEPC) in the presence of various cosolvents, the denaturation kinetics as a function of temperature have been determined based on Arrhenius plots derived from transition state theory analysis over the temperature range from 3.5°C to 65°C. The experimental data for phm-MDH were obtained in the presence of 1 M concentrations of various salts of monovalent and polyvalent anions, 1 M amino acids or 1 M sucrose and 6.1 M glycerol. Similarly, Arrhenius plot data were obtained for ml-PEPC in the presence of 2.5 M NaOAc and 0.8 M sodium glutamate. Distinct regimes of inactivation corresponding to high and low values of inactivation enthalpy were identified for the phm-MDH in the presence of all cosolvents and for the ml-PEPC in the presence of 2.5 M NaOAc, but not in the presence of 0.8 M sodium glutamate. A significant temperature-dependent effect dominated the inactivation of phm-MDH and ml-PEPC at elevated temperatures (e.g., ≥45°C), whilst the inactivation of these enzymes over a lower temperature range (≤25°C) was dominated by temperature-independent phenomenon. The corresponding thermodynamic activation parameters ( Δ G ‡, Δ H ‡ and Δ S ‡) associated with the transition state complexes involved in the inactivation of phm-MDH and ml-PEPC in the presence of the various cosolvents have been determined. The results indicate that the transition states associated with the inactivation of these two enzymes at elevated temperatures are characterised by large, positive enthalpic and entropic changes. In contrast, the inactivation process observed for phm-MDH at low temperatures in the presence of various cosolvents was marked by a large, negative entropic contribution and a small, positive enthalpic contribution. The results obtained in this study indicate that more than one mechanism of inactivation can occur with these two multimeric enzymes depending on the selected temperature range and the type of cosolvent. The relationship of these results to stabilisation models for phm-MDH and ml-PEPC in the presence of various cosolvents, as well as the application of Arrhenius plot data to extrapolate the long term solution stability of these enzymes at lower temperatures from the pseudo-first order rate constants of inactivation experimentally derived over a range of temperatures, are discussed.

Stability studies on pig heart mitochondrial malate dehydrogenase: the effect of salts and amino acids

The effect of different salts and amino acids on the thermal stability and quaternary conformation of pig heart mitochondrial malate dehydrogenase ( phm-MDH) in solution has been determined. The effectiveness of salts of anions in the stabilisation of phm-MDH followed the order: Citrate > SO 4 2− ≥ Tartrate > Phosphate > F −, CH 3COO −> Cl −> Br −. Anions above and including Cl − in this series were increasingly effective in stabilising phm-MDH with a rise in salt concentration from 0.05 – 2 M, whilst Br − was destabilising under similar conditions. The effect of potassium salts of acetate, chloride and bromide at a concentration of 1 M on the quaternary conformation of phm-MDH correlated also with the relative order of anion stabilisation above, with the anions higher in the series increasingly promoting the formation of the dimeric conformation of the enzyme. The cations of the corresponding salts had a relatively neutral (Cs +, K +, Na +, (CH 3) 4N +, NH 4 +) to a destabilising ((CH 3) 4N +, NH 4 +, Li +) effect on phm-MDH. Potassium ferrocyanide and potassium ferricyanide conferred complex, concentration dependent effects on the stability of phm-MDH, unlike the salts described above. Salts of amino acids were effective in the stabilisation of phm-MDH against temperature induced changes, following the order: NaGlutamatec ≅ NaAspartate > NaGlycinate > lysine · HCl > arginine · HCl. The magnitudes and trends of the effects of these salts and amino acids on the stability and quaternary structure of phm-MDH were observed to correlate well with considerations based on the Hofmeister series of anions and solvophobic concepts as they apply to the influence of co-solvents at intermediate to higher concentrations. Other, more specific effects were also evident in the stabilisation and destabilisation of phm-MDH by low concentrations of the salts, as noted most particularly in the presence of potassium ferrocyanide and potassium ferricyanide.

Reactivity of essential cysteine and lysine residues present at the catalytic domain of pig heart mitochondrial malate dehydrogenase.

Pig heart mitochondrial malate dehydrogenase was inactivated very rapidly by omicron-phthalaldehyde as compared to 5,5'-dithio bis(2-nitrobenzoic acid) and pyridoxal 5'-phosphate. The omicron-phthalaldehyde reaction followed pseudo first order kinetics, and a second order rate constant of 38 M-1 S-1 was obtained. Cysteine and lysine residues participating in the omicron-phthalaldehyde reaction are located in the NADH binding region of malate dehydrogenase as shown by protection experiments. The decrease in the rate of inactivation in the presence of NADH was used to determine the dissociation constant of the enzyme-NADH complex. pH dependent studies and molar transition energy calculations of the omicron-phthalaldehyde-inactivated enzyme have indicated that cysteine and lysine residues involved in the isoindole derivative formation are located in a hydrophobic environment at the coenzyme binding site.

Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES

Pig heart mitochondrial malate dehydrogenase was chemically denatured in guanidine HCl. Upon 50-fold dilution of the denaturant spontaneous refolding could be observed in the temperature range 12–32°C. At 36°C spontaneous refolding was not observed but a stable folding intermediate that is fairly resistant to aggregation was formed. This intermediate is readily refolded by the chaperonins GroEL and GroES and may prove useful in future attempts to describe several aspects of chaperonin action at physiological temperatures.

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 dehydrogenasesynthase 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 β-lactoglobulin.

Quantitation of the interaction between citrate synthase and malate dehydrogenase.

Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.

A facile method for the isolation of porcine heart mitochondrial malate dehydrogenase by affinity elution chromatography on Procion Red HE3B.

A quick, simple method has been devised for isolating pig heart mitochondrial malate dehydrogenase in apparently homogeneous state and good yield. It entails the adsorption of the enzyme to agarose-linked Procion Red HE3B and specific elution of a ternary complex consisting of the malate dehydrogenase, NAD+, and L-malate.

The effect of ionic environment on pig heart mitochondrial malate dehydrogenase

1. 1. The effect of ionic environment on pig heart mitochondrial malate dehydrogenase was investigated by means of two low resolution conformational probes, thermal stability and proteolytic lability. 2. 2. High ionic strength and high enzyme concentrations stabilize the enzyme towards thermal inactivation, probably by maintaining the enzyme in the dimeric form. 3. 3. Proteolysis of malate dehydrogenase under conditions where the enzyme is thermally stable indicates a secondary ionic effect on structure. 4. 4. Attempts to demonstrate dissociation of native malate dehydrogenase dimer into its constituent subunits using gel filtration on Sephacryl S-200 proved inconclusive. At low ionic strength malate dehydrogenase does not exhibit normal gel nitration behaviour.

Affinity labeling of a lysine residue in the coenzyme binding site of pig heart mitochondrial malate dehydrogenase

Affinity labeling of a lysine residue in the coenzyme binding site of pig heart mitochondrial malate dehydrogenase

Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies Homogeneous enzyme immunoassay for thryroxine

Pig heart mitochondrial malate dehydrogenase ( l-malate:NAD + oxidoreductase, EC 1.1.1.37) is about 90% inhibited upon labelling an average of two amino groups per subunit with an active ester of thyroxine. Inhibition is probably associated primarily with thyroxine binding to one specific group which is normally unreactive but becomes activated upon noncovalent binding of thyroxine derivatives to the enzyme. Enzyme inhibition is due to a decrease in the rate of association of NAD. Antibodies to thyroxine induce a slow conformational change with partial reversal of inhibition of more heavily labelled conjugates. The antibody-induced activation is not cooperative and does not require bivalent association of the antibody. Activation can be blocked by the presence of free thyroxine and is the basis for a clinically useful assay for serum thyroxine.

Inhibition of malate dehydrogenase by thyroxine and structurally related compounds

The inhibition of pig heart mitochondrial malate dehydrogenase ( l-malate: NAD + oxidoreductase, EC 1.1.1.37) by thyroxine and structurally related compounds was studied to resolve a longstanding question about the exact nature of the inhibition. Thyroxine, in freshly prepared solution, was found to be a “pure” conpetitive inhibitor relative to the nucleotide cofactor. Upon standing in diffuse daylight, solutions of thyroxine showed increased ability to inhibit the enzyme, presumably as a result of oxidation of enzyme sulfhydryl groups by free iodine that is released photochemically. This behavior probably accounts for earlier reports of irreversible inactivation by thyroxine. Comment is made on the implications of these findings to the mechanism of thyroid hormone action.

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.

Effect of Lipids on Enzymatic Activity of Pig Heart Mitochondrial Malate Dehydrogenase Monomolecular Films

The effect of various lipids on the enzymatic activity of pig heart mitochondrial malate dehydrogenase monomolecular films was studied using the subphase exchange technique described previously. Surface pressure-surface area (π–A) curves of mixed films of the enzyme with dipalmitoyllecithin, egg lecithin, cholesterol, and phospholipids extracted from pig hearts showed that the enzyme interacted with all of the lipids and that the enzyme remained in the film at pressures well above the collapse pressure of malate dehydrogenase in the absence of lipid. The surface enzyme activity was dependent on surface pressure for each lipid; in all cases, the lipids greatly broadened the range of surface pressures where surface enzyme activity was observed. The π–A and enzyme activity data showed good correlation. Although the simple model system employed does not simulate the complexity of the biological membrane, it gives some evidence for the role of lipids in the stability of membrane-bound enzymes.

Enzymatic activity of pig heart mitochondrial malate dehydrogenase monomolecular films by surface exchange technique.

A technique for studying the catalytic activity of enzymes spread as a film at an air-water interface, by exchanging the subphase under the film to remove unspread enzyme molecules, was developed, and its effectiveness was studied using surface-spread mitochondrial malate dehydrogenase. Mitochondrial malate dehydrogenase formed stable films which gave reproducible pi-A curves. The enzyme activity was measured by the oxidation rate of reduced nicotinamide adenine dinucleotide (NADH) in the presence of the substrate oxalacetic acid. Oxalacetic acid and NADH were injected into the subphase. The catalytic activity of the enzyme was dependent on the surface pressure of the film. The maximum catalytic activity was observed at a surface pressure of 4.4 dynes/cm. The activity was higher at intermediate surface pressures than at very low or very high surface pressures. A high bulk catalytic activity was observed in the unstable region, i.e., at a high degree of compression, of the film. The catalytic activity of the surface-spread enzyme was only a fraction of an equivalent amount of enzyme in solution.

Malate dehydrogenase: A higher molecular weight form produced by freeze-thaw treatment of pig heart supernatant enzyme

Attempts to produce hybrids of pig heart supernatant and mitochondrial malate dehydrogenase with the use of guanidine hydrochloride, acid treatment, and freezethaw techniques have been unsuccessful. However, the freeze-thaw technique produced a catalytically active higher molecular weight form of supernatant malate dehydrogenase in the absence or presence of mitochondrial enzyme. The higher molecular weight of this artifact was established by gel filtration and gel electrophoresis criteria. The specific activity of the artifactual form of the enzyme appears to be close to that of native supernatant malate dehydrogenase.

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5′-phosphate

1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.

Malate dehydrogenase: XI. Absence of tryptophan in porcine heart mitochondrial enzyme

Native and guanidine hydrochloride-denatured pig heart mitochondrial malate dehydrogenase have fluorescence and phosphorescence emission spectra resembling tyrosine but not that of tryptophan. Furthermore, amino acid analysis of acid and alkaline hydrolysates of the pure enzyme, designed to detect 0.15 tryptophan residues or more per subunit, failed to show detectable quantities. It is confirmed that the enzyme lacks tryptophan and this amino acid can have no function in the catalytic mechanism.

Identification of an essential, reactive histidine in pig heart mitochondrial malate dehydrogenase.

1 Iodoacetamide was found to inactivate pig heart mitochondrial malate dehydrogenase in a pseudo first order reaction. 2 Two moles of 14C-labelled reagent per mole of enzyme are incorporated into the protein. 3 The reactive residue was identified by amino acid analysis and thin layer chromatography as a histidine alkylated at nitrogen 3.

A reactive histidine residue at the active site of pig heart mitochondrial malate dehydrogenase.

A reactive histidine residue at the active site of pig heart mitochondrial malate dehydrogenase.