Malate dehydrogenase isozymes in the longnose dace,Rhinichthys cataractae

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

Purification of Chlorella Malate Dehydrogenase

Our prior investigation of Chlorella malate dehydrogenase (MDH) revealed supernatant and particulate isoenzymes which were immunologically, chromatographically, and electro-phoretically distinct. By means of ammonium sulfate fractionation and column chromatography a crystalline preparation of the particulate isoenzyme, specific activity of approximately 2000 has been obtained. It appears homogeneous in the analytical ultracentrifuge, by column chromatography and by electrophoresis. This preparation migrates as a single band of activity when subjected to starch gel electrophoresis and its mobility and activity (on the gel) are unaffected by prior incubation with citric acid, pH 2.0, EDTA, β-mercapto-ethanol, substrates or products; however, new bands appear when preincubated with exogenous proteins (RNase, Ovalbumin, Aldolase).     

Isozyme expression in F1 hybrids between carp and goldfish

Interspecific genetic differences in malate dehydrogenase (MDH), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and esterase (EST) isozymes in carp (Cyprinus carpio) and goldfish (Carassius auratus) were used to examine the allelic expressions in the hybrid between these species. A unique liver SOD and muscle LDH phenotype unambiguously identifies all presumed hybrid individuals. There was no evidence of F₂ or backcross phenotypes in hybrid individuals. Liver MDH and EST phenotypes in hybrids show a preferential expression of goldfish isozymes. Variation in the levels of carp liver MDH isozymes may result from the polymorphism of a regulatory mutation affecting isozyme expression, leading to gene silencing after duplication.This work was supported through NSERC (Canada) grants to James P. Bogart and John F. Leatherland.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Polymorphism of malate dehydrogenase in Ascaris suum

Starch gel electrophoresis of homogenates prepared from adult Ascaris suum revealed polymorphism for the number, staining intensity, and electrophoretic mobility of the cytoplasmic isozymes of malate dehydrogenase (MDH). Five different variant isozymic patterns were found among the 2160 worms surveyed. The most acceptable formulation for the molecular basis of the variant patterns supports the hypothesis that the synthesis of supernatant MDH in Ascaris suum is under the control of two separate genetic loci, MDH _A and MDH _B.This work was supported by National Institutes of Health Grant HD-00994.     

Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

Esterase and Malate Dehydrogenase Phenotypes in Portuguese Populations of Meloidogyne Species

Nonspecific esterases and malate dehydrogenases of 1-5 females from 40 root-knot nematode populations from Portugal were analyzed by electrophoresis in 0.4-mm-thick polyacrylamide gels. Fourteen major bands of esterase activity were detected, corresponding to 10 distinct phenotypes, Meloidogyne javanica and M. hapla had distinct species-specific phenotypes. Two phenotypes occurred in M. arenaria. The most variability was found among M. incognita populations. Of the remaining two phenotypes, one was associated with M. hispanica and the other belonged to a new species. Three malate dehydrogenase phenotypes were discerned on the basis of particular combinations of the eight main bands of activity found. As previously found, esterases were more useful than malate dehydrogenases in identification of the major Meloidogyne species. The host plant had no effect on the nematode esterase or malate dehydrogenase phenotypes.     

Genetics and polymorphism of a duplicated malate dehydrogenase (MDH) locus in the grasshopperOxya japonica japonica (Orthoptera:Acrididae:Oxyinae)

A biochemical genetic study of the enzyme malate dehydrogenase (MDH) was conducted in the grasshopperOxya j. japonica. Analysis of MDH electrophoretic variation in this species of grasshopper shows that one of the two autosomal loci for MDH in grasshoppers, the Mdh-2 locus, controlling the anodal set of MDH isozymes, is duplicated. Results of breeding studies confirm this and the observed polymorphism at theMdh-2 locus in the two populations ofOxya j. japonica studied can be attributed to three forms of linked alleles at the duplicated locus in equilibrium in both populations. In this respect, all individuals of this species possess heterozygous allelic combinations at the duplicatedMdh-2 locus, which may account for the spread of the duplicated locus in the populations of this species of grasshopper.This research was supported by a grant (Vote F) from the University of Malaya, Kuala Lumpur.     

4,4'-Bis dimethylaminodiphenylcarbinol: a new reagent for selective chemical modification. Interaction with porcine malate dehydrogenase

4,4-bis Dimethylaminodiphenylcarbinol (BDC-OH) has recently been reported to be a highly sensitive reagent for the quantitative determination of sulfhydryl residues in biological materials (1). In this communication the effectiveness of BDC-OH as a reagent for selective chemical modification of “active center” cysteine residues was investigated. The supernatant and mitochondrial forms of malate dehydrogenase were chosen for investigation by this reagent. Supernatant malate dehydrogenase which has never been found to contain an “active center” cysteine is unaffected by this reagent. Mitochondrial malate dehydrogenase (L malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart can be irreversibly inactivated by a 20 fold M excess of the reagent. Chemical modification of two essential sulfhydryl residues is prevented by the presence of the coenzyme, NAD⁺, suggesting that the site of interaction is located at or near the coenzyme binding site and hence at or near the enzymatic center of this enzyme.     

Enzymes of Carbohydrate Metabolism in Four Human Species of Leishmania: A Comparative Survey*

SYNOPSIS. The occurrence and levels of activity of various enzymes of carbohydrate catabolism in culture forms (promastigotes) of 4 human species of Leishmania (L. brasiliensis, L. donovani, L. mexicana, and L. tropica) were compared. These organisms possess enzymes of the Embden-Meyerhof pathway but lack lactate dehydrogenase. No evidence could be found for the production of lactic acid by growing cultures and lactic acid could not be detected either in cell-free preparations or after incubation of cell-free extracts with pyruvate and NADH under appropriate conditions. All 4 species possess α-glycerophosphate dehydrogenase and α-glycerophosphate phosphatase which together could regenerate NAD, thus compensating for the absence of lactate dehydrogenase. The oxidative and nonoxidative reactions of the hexose monophosphate pathway are present in all 4 species. Cell-free extracts have pyruvate dehydrogenase activity which allows the entry of pyruvate into and its subsequent oxidation through the tricarboxylic acid cycle. All enzymes of this cycle, including a thiamine pyrophosphate dependent α-ketoglutarate dehydrogenase are present. Both NAD and NADP-linked malate dehydrogenase activities are present. The isocitrate dehydrogenase is NADP specific. There is an active glutamate dehydrogenase which could compete with α-ketoglutarate dehydrogenase for the common substrate (α-ketoglutarate). Replenishment of C₄ acids is accomplished by heterotrophic CO₂ fixation catalyzed by pyruvate carboxylase. All 4 species have high levels of NADH oxidase activity. Several enzymes thus far not found in any species of Leishmania have been demonstrated. These are: phosphoglucose isomerase, triose phosphate isomerase, fructose-1, 6-diphosphatase, 3-phosphoglycerate kinase, enolase, α-glycerophosphate dehydrogenase, α-glycerophosphate phosphatase, pyruvate dehydrogenase complex, citrate synthase, aconitase, α-ketoglutarate dehydrogenase, glutamate dehydrogenase, and NADH oxidase.     

Electrophoretic variation of supernatant malate dehydrogenase in marsupials

Starch gel electrophoresis of supernatant malate dehydrogenase (MDH A₂) was performed on erythrocyte samples from 505 individual animals representative of 33 marsupial species. Most species exhibited electrophoretically identical forms of MDH A₂ activity with the exception of the grey kangaroos, Trichosurus possums, and bandicoots, thus confirming the phylogenetic relatedness of animals within each group and the conservative nature of this enzyme. Polymorphisms were observed in two of the six species analyzed whose mobilities were non-standard. Allelic isozyme patterns and those from interspecies F₁ hybrids between grey kangaroos and other macropods were consistent with a dimeric subunit structure and an autosomal locus (MDH-A) encoding the enzyme.Supported in part by grants from the Australian Research Grants Committee.     

Leaf-eating invertebrates as competitors of aquatic hyphomycetes

Summary Leaf-eating invertebrates selectively ingest leaf areas rich in fungal cells. The effect of this process on coincident and cumulative species diversity (species numbers and evenness) of the fungi was studied on 3 substrates (oak leaves, larch and spruce needles) in 2 hardwater and 2 softwater streams. Cumulative species number of colonizing fungi follows the equation S=k·A ^z(A=area below decay curve of the substrate, k=substrate-specific constant, Z=0.47). Higher feeding activity means faster weight loss of the substrate which leads to lower species richness of the fungi. The opposite is true for early successional stages on larch needles. Evenness of the fungi (distribution of individuals among species) is negatively correlated with feeding intensity by invertebrates, as measured by increased decay rates. The overall effect of leaf-eating invertebrates on aquatic hyphomycetes resembles that of potent competitors preempting substrate otherwise used by a late successional tail of relatively rare fungi.     

Enzymatic Activity in Mitochondria from Orobanche

Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence.     

Schistosoma mansoni: antigenic characterization of malate dehydrogenase isoenzymes and use in the defined antigen substrate spheres (DASS) system.

Immunoelectrophoresis of Schistosoma mansoni homogenates against mouse antisera resulted in only one precipitation line, which showed malate dehydrogenase activity. Immunoprecipitins against schistosomal malate dehydrogenase were also demonstrated in sera from individuals with schistosomiasis. Analysis by the double-diffusion method showed that malate dehydrogenase antigens in S. mansoni, S. haematobium, and S. bovis are immunologically indistinguishable. Immunoelectrophoresis of isolated mitochondrial and cytoplasmic malate dehydrogenase, showed that only the mitochondrial enzyme is able to form a malate dehydrogenase active precipitation line. Rabbit antisera directed against purified mitochondrial malate dehydrogenase showed a reaction with the enzyme as judge by immunoelectrophoresis. A purified mitochondrial malate dehydrogenase preparation, coupled to Sepharose 4B, was used in the defined antigen substrate spheres (DASS) test. Sera from experimentally infected mice contained considerably higher levels of antibodies against the mitochondrial malate dehydrogenase preparation than sera from infected individuals.     

Genetics of human-mouse somatic cell hybrids: Linkage of human genes for isocitrate dehydrogenase and malate dehydrogenase

Based on somatic cell genetic analysis, autosomal gene linkage is reported for the supernatant enzymes of human isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) in human-mouse cell hybrids. The IDH, MDH linkage was not linked to the X and E ₁₇ chromosomes or to 12 additional human enzyme markers.This work was supported in part by grants from the U.S. Public Health Service (Child Health and Human Development) and the United Health Foundation of Western New York.     

Detection of an NAD+-dependent malic enzyme locus in the atlantic salmonSalmo salar and other salmonid fish

Electrophoretic studies of malate oxidoreductases routinely assess variation in two enzymes, malate dehydrogenase (EC 1.1.1.37) and malic enzyme (NADP⁺) (EC 1.1.1.40). By modification of the standard isozyme staining conditions for these enzymes, we have resolved a new NAD⁺-preferring, MgCl₂-requiring malic enzyme which is indicated to be EC 1.1.1.39. The enzyme was detected in 10 salmonid fish species of the generaSalmo, Salvelinus, andOnchoryhncus. Phenotypic variation indicates that the novel enzyme is tetrameric and coded by a single locus. Inheritance inS. salar follows a single-locus model and the phenotypes are unlinked to polymorphisms for_s MDH-3,4* and_m MEP-2*, two malate oxidoreductase loci previously shown to be variable in this species.This work was supported by a contract to E. V. from Fisheries and Oceans Canada, St. John's, Newfoundland, and a postgraduate award to W. C. J. from the Department of Education for Northern Ireland.     

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

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. The tissue distribution of s-MDH and ADH isozymes in both species is consistent with spatial profiles reported for other teleosts. The expression of alleles of paternal origin at the s-Mdh-B and Adh loci 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.     

Enzymes of carbohydrate metabolism ofCotugnia digonopora and their activity in the presence of anthelmintics,in vitro

Cotugnia digonopora, a fowl cycllophyllidean cestode, was found to possess most of the enzymes, associated with the glycolytic sequence and phosphoenolpyruvate branch point, in the cytosol fraction. Enzymes of malate metabolism were predominantly mitrochondrial. Anthelmintic agents inhibited hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, fumarate reductase, and malic enzyme. In intact worms this effect was significantly reduced. However, the activities of glycogen Phosphorylase and pyruvate kinase were significantly enhanced. Communication No. 4113 from CDRI, Lucknow.     

The effect of leaf pack composition on processing: A comparison of mixed and single species packs

The effect of leaf species composition on decomposition patterns was examined in a coastal plain stream. Red maple leaves (Acer rubrum) decomposed at the same rate separately or when mixed with cypress leaves (Taxodium distichum). Cypress addition increased structural integrity but its effects differed between sites with different hydrologic regimes. Invertebrate communities varied slightly between mixed and single species packs, however invertebrates did not appear to be the primary agent of decomposition. Mixed species packs may be an alternative method to fine mesh bags for studying processing of small, narrow leaves in a more realistic manner.     

AN ELECTROPHORETIC AND SEROLOGICAL INVESTIGATION OF SEED PROTEINS IN GALEOPSIS TETRAHIT L. (LABIATAE) AND ITS PUTATIVE PARENTAL SPECIES

Seed proteins from the tetraploid Galeopsis tetrahit L. and its putative parental species G. pubescens Bess. and G. speciosa Mill., were examined using disc gel electrophoresis (PAGE), Ouchterlony double diffusion, and Immunoelectrophoresis. Forty-three inbred cultivated lines of G. speciosa, G. pubescens, and G. tetrahit (both the naturally occurring species and an artificially produced hybrid) and two wild populations of G. tetrahit were examined. PAGE gels were stained for total protein or malate dehydrogenase. Although variability in the total protein pattern was detected within and between lines for all three species, there was much similarity among the taxa. No bands appeared in the hybrid that were not present in the parents. The band pattern for synthetic G. tetrahit was very similar to that of natural G. tetrahit suggesting that G. tetrahit originated from a cross in nature between G. pubescens and G. speciosa. The total protein PAGE results were analyzed using numerical taxonomic techniques. Although these analyses did not clearly separate the three species, the general tendency was toward grouping G. pubescens lines together, natural G. tetrahit lines together, and synthetic G. tetrahit lines together. Natural and synthetic G. tetrahit lines were also interspersed among each other along with some G. speciosa lines. The malate dehydrogenase pattern showed little variation among the three species. Qualitative serological methods did not distingush among the three species, but confirmed the similarity observed among the taxa in the results of electrophoresis.