Supernatant Malate Dehydrogenase Research Articles

Mor2, supernatant malate dehydrogenase, is linked to wa2 and Hba on mouse chromosome 11 in a region of homology with human chromosome 2p.

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized thewobblerspinal atrophy genewrto proximal mouse Chr 11, tightly linked toRab1,a gene coding for a small GTP-binding protein, andGlns-ps1,an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of theRab1region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescencein situhybridization (FISH), and sequence-tagged site (STS) isolation and mapping.Rab1andGlns-ps1were found to be only 200 kb apart. A potential CpG island near a methylatedNarI site and a trapped exon,ETG1.1,were found between these loci, and a new STS,AHY1.1,was found over 250 kb fromRab1.Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising theRAB1locus,AHY1.1,and the human homologue ofETG1.1,indicating a high degree of conservation of this region in the two species. We mappedAHY1.1and thus humanRAB1on Chr 2p13.4–p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the geneLMGMD2Bfor a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13–p16. The conservation between the mouseRab1and humanRAB1regions will be helpful in identifying candidate genes for thewobblerspinal muscular atrophy and in clarifying a possible relationship betweenwrandLMGMD2B.

Evaluation of Stocking Walleye Fry to Enhance a Commercial Fishery in a Large, Canadian Prairie Lake

Abstract More than 100 million fry of walleye Stizostedion vitreum are stocked annually into lakes of the prairie provinces of Canada to enhance sport and commercial fisheries. However, the success of this practice has not been evaluated. We introduced 7.1 million genetically marked walleye fry into Dauphin Lake, a 50,000-hectare lake in Manitoba, in 1985 to evaluate fry stocking as a tool for fishery enhancement. The 1985 walleye cohort was fully recruited into the commercial fishery at age 3 (in 1988). We sampled commercial catches in the winters of 1988 and 1989 to determine the phenotypes for supernatant malate dehydrogenase in 727 3-year-old walleyes and 657 4-year-old walleyes of the 1985 (enhanced) year-class. The introduced fish contributed about 2.9% to the 1985 year-class of walleyes. The survival of walleye fry from the time of stocking to age 3 (in the fall) was estimated at 0.04%. At current walleye harvest rates in Dauphin Lake (about 13,000 kg/year), the stocked fish were expected to contri...

Asynchronous expression of the alcohol and supernatant malate dehydrogenase loci during Barbus hybrid development (Cypriniformes, Teleostei)

1.1. The tissue specificity and ontogeny of supernatant malate dehydrogenase (s-MDH) and alcohol dehydrogenase (ADH) are reported for the tiger barb (Barbus tetrazona), the rosy barb (Barbus conchonius) and their reciprocal hybrids.2.2. The tissue distribution of s-MDH and ADH isozymes in both species is consistent with spatial profiles reported for other teleosts.3.3. The expression of alleles of paternal origin at the s-Mdh-B and Adhloci are delayed in reciprocal hybrids as compared to their expression intraspecifically; suggestive of a low degree of affinity between maternally derived regulatory factors and paternal regulative elements controlling structural gene activation.

Comparison of the spatial and temporal expression of supernatant malate dehydrogenase in Barbus hybrids (Cypriniformes, teleostei)

1.1. The tissue specificity and ontogeny of supernatant malate dehydrogenase (s-MDH) are reported for the tiger barb, cherry barb, and their reciprocal hybrids.2.2. The tissue distribution of s-MDH isozymes in Barbus is consistent with the patterns reported in other teleosts.3.3. The expression of the Mdh-B locus is correlated with the initial muscle contractions of the developing embryos. It is suggested that the state of muscle cell differentiation may be the stimulus necessary for the expression of this locus in Barbus.4.4. Expression of maternal and paternal alleles at the B locus are synchronously delayed in reciprocal hybrids, as compared to their expression intraspecifically.

THE DEVELOPMENTAL APPEARANCE OF PATERNAL FORMS OF LACTATE DEHYDROGENASE AND MALATE DEHYDROGENASE IN HYBRID HORSESHOE CRABS.

Differences in electrophoretic mobilities of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) existed between three Asian horseshoe crabs, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda, used for interspecific hybridization. After electrophoresis of extracts of hybrid horseshoe crab embryos on starch gels, the paternal, maternal, and hybrid forms of the LDH and MDH were detected with specific enzyme staining. In viable hybrids the paternal form of the LDH was detected at stage 17 (immediately before the 1st embryonic molt). Similarly, evidence of gene expression for mitochondrial MDH was seen at stage 14 (stage of appearance of rudimental appendages). Gene expression for supernatant MDH was seen at stage 17 (immediately before the 1st embryonic molt). Regarding the onset of genome control in embryogenesis, it was suggested that prior to the activation of the maternal gene of the LDH, the paternal gene of the LDH was activated in horseshoe crab hybrids. Furthermore, there was evidence that the maternal effects on early embryogenesis were due to enzymes present in the egg prior to fertilization, not to continued synthesis directed by stable messenger RNA.

Adaptative features of ectothermic enzymes—IV. Studies on malate dehydrogenase of Astyanax fasciatus (Characidae) from lobo reservoir (São Carlos, São Paulo, Brasil)

1. 1. Skeletal muscle and heart supernatant malate dehydrogenase (s-MDH) from a subtropical fish, Astyanax fasciatus consists of three electrophoretically anodal bands. Each band is a dimer (AA, AB and BB) and two loci are active. 2. 2. In A. fasciatus tissue extracts, A and B subunits are present at differing quantitative levels and their activities are almost season-independent. However, the relative activity of each homodimer in relation to total s-MDH estimated by densitometry of gels or of each homodimer purified by chromatography varies with temperature. The more anodic homodimer is termolabile and the less anodic one is thermostable. 3. 3. The pH optimum of s-MDH is 7.5, of AA is 6.5 and of BB is 7.8. 4. 4. The BB isozyme is more sensitive to high concentrations of substrate and has a K m temperature-independent. The AA isozyme is not inhibited by high concentrations of oxaloacetate and shows a K m temperature-dependent with a fourteenfold increase between 20° and 40°C.

Increased Heterozygosity at the Mdh-B Locus in Fish Inhabiting a Rapidly Fluctuating Thermal Environment

Populations of a common forage fish, red shiner Notropis lutrensis, were sampled from four localities on the Brazos River, Texas, affected by cold-water discharge from a hydroelectric dam and from unaltered sites in the same region. Polymorphism at the Mdh-B locus, encoding supernatant malate dehydrogenase, indicates that populations within 57 km of the dam are distinctive from other regional populations and possess a unique Mdh-B allele, have significantly higher levels of heterozygosity at the Mdh-B locus, represent a homogeneous set that have significantly different Mdh-B zygotic frequencies from other regional populations, and have significantly different Mdh-B zygotic proportions than would be expected under a Hardy-Weinberg equilibrium. Increased levels of heterozygosity in fish within 57 km of the dam were correlated with discharge-associated fluctuations in water temperature at sampling stations.

The oxaloacetate reductase activity of vertebrate lactate dehydrogenase

1. 1. Lactate dehydrogenase is able to catalyse the reduction of oxaloacetate utilizing NADH as coenzyme but, contrary to a previous report, with greatly reduced values for both K m and V max when compared to the normal substrate pyruvate. 2. 2. A modification to the published procedure for the purification of vertebrate l-lactate dehydrogenase by affinity chromatography on oxamated Sepharose is described. 3. 3. Supernatant malate dehydrogenase does not catalyse the reduction of pyruvate at rates greater than 1%, of the rates with oxaloacetate.

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.

Binding of reduced and oxidized nicotinamide adenine dinucleotide to pig heart supernatant malate dehydrogenase

The association reactions of NADH and NAD+ with dimeric pig heart supernatant malate dehydrogenase (s-MDH) have been measured at pH 6 and 8 by calorimetric and fluorescence methods, and the thermodynamic parameters describing these reactions have been evaluated. Coenzyme binding is associated with the uptake of 0.55 mol of H+/mol of NADH at pH 8 and 0.19 mol of H+ at pH 6. No significant effect of NAD+ binding on proton binding was observed. Increase in ionic strength strongly affects the free energies of binding of NAD+ and NADH. No cooperativity was observed in the enthalpy or free energy changes for binding of NAD+ or NADH. The differences in free energy of binding of NAD+ and NADH and the effect of pH on binding of NADH are entropy based. These effects are interpreted as reflecting a small number of interactions within the active site that are predominantly ionic.

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10 −6, M −1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by V H V D and ( V H K H ) V D K D values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.

Malate dehydrogenase, inhibition of pig heart supernatant enzyme by iodoacetamide.

Pig heart supernatant malate dehydrogenase is alkylated by 250 mM iodoacetamide at 37 degrees C in pH 7.5 Tris/acetate buffer which is 0.05 M with respect to acetate to form enzyme with 1,3-dicarboxamidomethyl histidine, 3-carboxamidomethyl histidine, 1-carboxamidomethyl histidine, carboxamidomethyl cysteine, and carboxamidomethyl methionine. 1,3-Dicarboxamidomethyl histidine forms with a stoichiometry of 1/enzyme subunit at a rate equaling the rate (approximately 1.24 +/- 0.09 X 10(-2) min-1) of enzyme inactivation by iodoacetamide. All other amino acid derivatives form at a slower rate initially or continuously. The enzyme is protected against inactivation by iodoacetamide through NADH binding, suggesting that there is an "essential" histidine residue at or near the active site.

Malate dehydrogenase. Kinetic studies of substrate activation of supernatant enzyme by L-malate

At pH 8.0 in 0.05 M Tris-acetate buffer at 25 degrees C, homogeneous supernatant malate dehydrogenase exhibits substrate activation by L-malate. The turnover number, Michaelis constant for L-malate, and Michaelis constant for NAD are: 0.46 X 10(4) min(-1), 0.036 mM, and 0.14 mM, respectively, for nonactivated enzyme and 1.1 X 10(4) min(-1), 0.2mM, and 0.047 mM for the same series of constants in activated enzyme. Nonactivating behavior is observed at concentrations between 0.02 and 0.15 mM L-malate and activating behavior is observed between 0.15 and 0.5 mM L-malate. L-Malate activation is compared with similar activation of mitochondrial malate dehydrogenase. While it is not possible to exclude unequivocally all mechanisms, the data seem to be consistent with the occurrence of a fundamentally ordered bi bi mechanism, possibly involving activation through the allosteric binding of L-malate. It is concluded that the data are consistent with a form of the "reciprocating compulsory order mechanism" in which nonactivated enzyme reflects catalysis by one subunit and activated catalysis expresses the coordinated activity of two subunits. The allosteric interaction and the "reciprocating mechanism/ are not mutually exclusive.

Effect of temperature on activity of allozymic forms of supernatant malate dehydrogenase in the red shiner, Notropis lutrensis

1. 1. Electrophoretic patterns for supernatant malate dehydrogenase in a common forage fish, Notropis lutrensis, were observed for several populations. 2. 2. The patterns observed result from duplication of the supernatant MDH locus resulting in three isozymes. Genetic polymorphism results in three distinct allozymic patterns. Heterozygous combinations between any two allozymic forms results in six isozymes. 3. 3. Thermal studies of enzyme activity revealed that cathodal isozymes of the three allozymic forms have different temperature-related optima, i.e. 20, 25 and 30°C.

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.

Fasciola hepatica: The subcellular distribution and kinetic and electrophoretic properties of malate dehydrogenase

The optimal pH for oxalacetate reduction was 9.0 for the mitochondrial and supernatant enzyme. The optimal pH for malate oxidation was 10 for both fractions. Maximal activity during oxalacetate reduction was 3.26 ± 0.26 μmoles of NADH oxidized/mg of protein/min and 3.02 ± 0.27 for the supernatant and mitochondrial fractions, respectively. Maximal malate oxidation was 0.495 ± 0.08 and 0.72 ± 0.12 (not statistically different) for the supernatant and mitochondrial fractions, respectively. High concentrations of malate inhibited activity to a greater extent in the supernatant fraction while oxalacetate inhibited activity to a greater extent in the mitochondrial fraction. The supernatant malate dehydrogenase was more heat stable than the mitochrondrial enzyme. Electrophoresis showed three isoenzymes of malate dehydrogenase in whole homogenates of Fasciola hepatica and all three were represented in the supernatant while only one band was seen in the mitochondrial fraction. The presence of two types of malate dehydrogenase, one in the supernatant with three isoenzymes and one in the mitochondria with one isoenzyme, is proposed.

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.

Comparative enzymology of malate dehydrogenases—VIII. Cytoplasmic characteristics of pig erythrocyte malate dehydrogenase

1. 1. Malate dehydrogenase (EC 1.1.1.37) was 100-fold purified from porcine erythrocytes, and a partially purified preparation, with specific activity of 3000–4200 U/mg, was obtained. Chromatographic and kinetic characteristics, and inhibition patterns, indicate that porcine erythrocytes possess a single isoenzyme of malate dehydrogenase with cytoplasmic, rather than mitochondrial characteristics. 2. 2. Partially purified erythrocyte malate dehydrogenase has the following limiting Michaelis constants for substrates: 56 gmM for NADH and 62 gmM for oxaloacetate at pH 7.6, and 135 gmM for NAD + and 1200 gmM for l-malate at pH 9.0, respectively. 3. 3. Molecular weight of the erythrocyte isoenzyme (70,000), estimated by the gel filtration method, is identical with the molecular weights of mitochondrial and supernatant malate dehydrogenase isoenzymes from pig heart. 4. 4. No meaningful metabolic pathway has been found which would connect the malate dehydrogenase reaction with the other metabolic functions of the erythrocyte. Therefore, the proposition has been advanced that the large quantity of malate dehydrogenase-activity (1-1.2 million U/l of whole blood) in erythrocytes, represents a functionless remainder after the dedifferentiation of reticulocytes into the mature red blood cells.

Inhibitory effect of bilirubin on bovine cerebral supernatant malate dehydrogenase activity

Inhibitory effect of bilirubin on bovine cerebral supernatant malate dehydrogenase activity

Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.