Molecular weight of cytoplasmic malate dehydrogenase, mitochondrial malate dehydrogenase and lactate dehydrogenase of a freshwater catfish

The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) were studied in the liver and skeletal muscle of the freshwater catfish, Clarias batrachus. There were two electrophoretically distinguishable bands (AA and BB) of cMDH and mMDH which suggests that they are apparently encoded at two gene loci (A and B) in both the tissues. However, the presence of a single band (LDH-1) of LDH in liver and double bands (LDH-1 and LDH-2) in skeletal muscle in which LDH-2 was predominant reflects the differential expression of LDH genes in different metabolic tissues to meet the requirement of energy production. The AA isoform (74 kd) of liver cMDH was smaller than those of the AA form (110 kd) of skeletal muscle. In contrast, the BB isoform of liver (42 kd) and skeletal muscle (54 kd) were more or less similar in size. Unlike the case of cMDH, the molecular weight of AA isoform (115 kd) of liver mMDH was higher than those of the AA form (87 kd) of skeletal muscle. Whereas the molecular weight of BB isoform (58 kd) of liver was in proximity to the weight of BB form (44 kd) of skeletal muscle mMDH. The size of AA isoform (74 kd) of liver cMDH was smaller, while the AA isoform (110 kd) of skeletal muscle was larger as compared to AA form of mMDH in the liver (115 kd) and skeletal muscle (87 kd). But the size of BB isoform of both the isozymes was almost equal in these metabolic tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical properties and chemical compositions of cytoplasmic and mitochondrial malate dehydrogenase from Physarum polycephalum

The malate dehydrogenase isoenzymes from Physarum polycephalum have been purified to homogeneity as confirmed by gel filtration chromatography, polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. Certain physical and chemical parameters of the malate dehydrogenase isoenzymes reported here include sedimentation, molecular weight and subunit molecular weight. Most unique of the differences between the isoenzymes were the widely separate isoelectric points of 9.83 for mitochondrial malate dehydrogenase and 6.14 for the supernatant malate dehydrogenase. The amino acid analyses of each form were done revealing the isoenzymes were unquestionably unique proteins differing in the content of ten amino acids.     

Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements

The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially-folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (phi = 18.6 ns) was determined for partially-folded species tagged with anthraniloyl. GroEL recognizes partially-folded species with a K(D) approximately 60 nM. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, approaches a value of 280 ns in the absence of ATP. Reactivation of mMDH-ANT by addition of GroEL and ATP brings about a significant decrease in the observed rotational correlation time. The results indicate that partially-folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.     

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.     

Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites. A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Biochemistry 35, 12652-12658], showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein. Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein. The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. This channeling behavior is eliminated at high ionic strength. A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate. A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.     

Lactate and malate dehydrogenase and alpha-esterases in oligospermia

Lactate dehydrogenase (LDH), malate dehydrogenase, and alpha-esterase were studied electrophoretically in a total of 99 semen samples obtained from normal, vasectomized, oligospermic, and infertile males. The enzymatic patterns were compared with total sperm count and percentage sperm motility. Lactate dehydrogenase X was absent in semen samples from oligospermic as well as vasectomized males. An extra LDH band (between the second and third LDH bands) was detectable in samples which had a low sperm count but higher motility (80 to 90%). This extra band was absent in samples with higher sperm count regardless of the level of percentage motility, suggesting that the extra band possibly may be related to motility factors of low sperm count but not normal sperm count. The fastest moving alpha-esterase bands were absent in samples from oligospermic or vasectomized males. These bands were also absent in samples from infertile patients having a very low sperm count. It is not clear whether the fast-moving esterase bands are related to sperm count or possible differences in hormonal levels of individuals with low or zero sperm count samples. The patterns of malate dehydrogenase did not differ in individuals with zero, low, or normal sperm count.     

Simulation of the enzyme reaction mechanism of malate dehydrogenase

A hybrid numerical method, which employs molecular mechanics to describe the bulk of the solvent-protein matrix and a semiempirical quantum-mechanical treatment for atoms near the reactive site, was utilized to simulate the minimum energy surface and reaction pathway for the interconversion of malate and oxaloacetate catalyzed by the enzyme malate dehydrogenase (MDH). A reaction mechanism for proton and hydride transfers associated with MDH and cofactor nicotinamide adenine dinucleotide (NAD) is deduced from the topology of the calculated energy surface. The proposed mechanism consists of (1) a sequential reaction with proton transfer preceding hydride transfer (malate to oxaloacetate direction), (2) the existence of two transition states with energy barriers of approximately 7 and 15 kcal/mol for the proton and hydride transfers, respectively, and (3) reactant (malate) and product (oxaloacetate) states that are nearly isoenergetic. Simulation analysis of the calculated energy profile shows that solvent effects due to the protein matrix dramatically alter the intrinsic reactivity of the functional groups involved in the MDH reaction, resulting in energetics similar to that found in aqueous solution. An energy decomposition analysis indicates that specific MDH residues (Arg-81, Arg-87, Asn-119, Asp-150, and Arg-153) in the vicinity of the substrate make significant energetic contributions to the stabilization of proton transfer and destabilization of hydride transfer. This suggests that these amino acids play an important role in the catalytic properties of MDH.     

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.     

Specific glucocorticoid binding at different levels of human motor activity

We studied the number of glucocorticoid receptors and dissociation constant in isolated human lymphocytes as well as blood concentrations of hormones produced by the hypothalamic-hypophyseal-adrenocortical system in three experimental series: at normal (17 subjects), decreased (10 subjects, a 360-d head-down bed rest) and increased (8 subjects, physical exercise on bicycle ergometer) levels of motor activity. In the first series we found that the number of glucocorticoid receptors and dissociation constant did not depend on the season, on the age of subjects nor on cortisol concentrations in blood. In the second series we observed the following: at the end of the first month of bed rest the number of glucocorticoid receptors and receptor affinity significantly increased; at the beginning of the third month of bed rest specific glucocorticoid binding significantly decreased and circadian rhythms of adrenocorticotropin and cortisol in blood varied markedly; at the end of the sixth month of bed rest the number of glucocorticoid receptors returned to prebed rest levels and dissociation constant decreased. In the third series physical exercises that induced an activation of the hypothalamic-hypophyseal-adrenocortical system (maximal physical exercises and prolonged submaximal exercises at 70% of maximal oxygen uptake) led to a significant increase in the number of glucocorticoid receptors without changes of dissociation constant. These results indicate that both a decrease and an increase of human motor activity resulted in significant changes of specific glucocorticoid binding which were not influenced by changes of circulating hormone concentrations in blood but by some other factors affected by physical activity.     

The autoinhibition of sorghum NADP malate dehydrogenase is mediated by a C-terminal negative charge

The chloroplastic NADP malate dehydrogenase is completely inactive in its oxidized form and is activated by thiol/disulfide interchange with reduced thioredoxin. To elucidate the molecular mechanism underlying the absence of activity of the oxidized enzyme, we used site-directed mutagenesis to delete or substitute the two most C-terminal residues (C-terminal Val, penultimate Glu, both bearing negative charges). We also combined these mutations with the elimination of one or both of the possible regulatory N-terminal disulfides by mutating the corresponding cysteines. Proteins mutated at the C-terminal residues had no activity in the oxidized form but were partially inhibited when pretreated with the histidine-specific reagent diethyl pyrocarbonate before activation, showing that the active site was partially accessible. Proteins missing both N-terminal regulatory disulfides reached almost full activity without activation upon elimination of the negative charge of the penultimate Glu. These results strongly support a model where the C-terminal extension is docked into the active site through a negatively charged residue, acting as an internal inhibitor. They show also that the reduction of both N-terminal bridges is necessary to release the C-terminal extension from the active site. This is the first report for a thiol-activated enzyme of a regulatory mechanism resembling the well known intrasteric inhibition of protein kinases.     

Purification and characterisation of isocitrate dehydrogenase and malate dehydrogenase from Mycobacterium tuberculosis and evaluation of their potential as suitable antigens for the serodiagnosis of tuberculosis

SETTING: Enzymes from Mycobacterium tuberculosis are potent antigens and might thus be of interest in the serodiagnosis of tuberculosis. OBJECTIVE: The purpose of the study was to purify and characterize the two enzymes isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) from, M. tuberculosis and to evaluate their potential in the serodiagnosis of tuberculosis. DESIGN: The two enzymes were analysed for specificity by electrophoresis and then purified by means of affinity chromatography using reactive dyes and ion exchange chromatography. The two isolated enzyme fractions were analysed by ELISA, using antisera against related organisms. They were then tested as antigens in ELISA together with sera from tuberculous patients and controls. RESULTS: The electrophoretical analyses showed that the two enzymes each differed markedly from the corresponding enzymes of other mycobacteria. The serological analyses, however, could not distinguish between either IDH or MDH from other mycobacteria, but organisms of other genera, such as Nocardia, gave much weaker responses. When IDH and MDH were tested with sera from tuberculous patients and controls the former gave clearly higher optical density values than the latter. CONCLUSION: The enzymes/antigens IDH and MDH may be of value in developing a serological test for tuberculosis. The latter fraction seemed particularly capable of discriminating patients from controls.