Purification and Properties of Chicken Heart Mitochondrial and Supernatant Malic Dehydrogenases<sup>*</sup>

Kitto, G. Barrie; Kaplan, Nathan O.

doi:10.1021/bi00876a031

Cited by 161 publications

(41 citation statements)

References 37 publications

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“…The recombinant protein displayed an identical pH profile with pH optima values identical to those obtained for the native enzyme in both directions. These observations correspond well with data obtained by other workers for m-MDH enzymes from different sources, in particular chicken heart m-MDH which displayed pH optima of 7.8 and 10 for oxaloacetate reduction and malate oxidation, respectively [56], while a pH optimum of 7.9 (oxaloacetate reduction) was determined for m-MDH from the marine snail, Ilyanassa obsoleta [57]. It is accepted that His195 is a key residue in the mechanism of MDH, and must be protonated for oxaloacetate binding and unprotonated for malate binding [58].…”

Section: Physicochemical Characterization Of T Emersonii Native and supporting

confidence: 92%

“…The K m value for the T. emersonii m-MDH with L-malate as substrate (1.25 mM; buffer A) is similar to data for m-MDHs from mammalian sources (e.g. 0.90 mM for chicken heart m-MDH [56]), but is markedly different to K m values for invertebrate and other fungal counterparts, e.g. 22.0 mM for the Mytilus edulis enzyme [31] and 0.25 mM for m-MDH from C. neoformans [36].…”

Section: Kinetic Characterization Of Native and Recombinant M-mdh Frosupporting

confidence: 71%

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Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Maloney

Callan

Murray

et al. 2004

European Journal of Biochemistry

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Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52°C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The fulllength m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni 2+ -nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.Keywords: malate dehydrogenase; filamentous fungus; bio-specific affinity chromatography; thermophilic eukaryote; Talaromyces emersonii.Malate dehydrogenase (MDH) catalyzes the pyridine nucleotide-dependent interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. It is also thought to have a role in the malate/aspartate shuttle across the inner mitochondrial membrane. In most eukaryotic cells there are two major isoenzymes of MDH, cytosolic MDH (c-MDH) and mitochondrial MDH (m-MDH) [1].The majority of m-MDHs isolated to date from eukaryotic sources are homodimers of identical subunits, with a monomer size of 34 kDa. Mitochondrial MDH displays a complex regulatory pattern that involves allosteric activation [2], membrane interaction [3] and formation of multienzyme complexes [4] with enzymes such as citrate synthase, aspartate aminotransferase and other mitochondrial components [5][6][7][8][9][10]. Much of the previous research has focused on the comparison of primary and three-dimensional structures of mitochondrial and cytoplasmic forms of MDH isolated from a single source, e.g. pig heart tissue [11,12]. In contrast to bacterial, vertebrate and invertebrate m-MDH, a paucity of information exists on fungal m-MDH with significantly more known about yeast m-MDH than homologs from filamentous fungal species. Previous research by Dalhaus et al. [13] and Goward and Nicholls [14] has proposed that investigation...

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Section: Physicochemical Characterization Of T Emersonii Native and supporting

confidence: 92%

Section: Kinetic Characterization Of Native and Recombinant M-mdh Frosupporting

confidence: 71%

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Maloney

Callan

Murray

et al. 2004

European Journal of Biochemistry

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“…For example, anti-chicken MDH-A2 precipitated or interacted with the three allelic isozymes migrating anodally while anti-chicken MDH-B2 treatment selectively removed the cathodal isozyme. This demonstrates the homologous relationships of these isozymes to those of the chicken, which have been fully characterized with respect to their subcellular location, subunit composition and genetic control (Kitto and Kaplan 1966). Similar immuno-chemical results have been previously reported by Grimm and Doherty (1961), who established the immunochemical specificities of these isozymes.…”

Section: Resultssupporting

confidence: 88%

“…Vertebrate malate dehydrogenase exists as two major isozymes, MDH-A2 and MDH-B2, which are localized in the cytoplasm and mitochondria respectively (Siegel and Englard 1961;Thorne et al 1963;Kitto and Kaplan 1966). Genetic and biochemical analyses have shown that these isozymes are coded by distinct nuclear genes (Shows et al 1970;Wheat et al 1971) and have dimeric subunit structures (Devenyi et al 1966;Kitto and Kaplan 1966;Courtner 1967a, 1967b).…”

Section: Introductionmentioning

confidence: 99%

Genetic Variation and Immunochemical Properties of L-Malate : NAD+ Oxidoreductase Isozymes in the Bandicoot, Isoodon macrourus.

Holmes¹,

VandeBerg²,

Gordeon³

1974

Aust. Jnl. Of Bio. Sci.

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A genetic polymorphism for the soluble isozyme (MDH-A2) of malate dehydrogenase (L-malate : NAD+ oxidoreductase; EC 1.1.1.37) was found in a Townsville-Inkerman population of short-nosed bandicoots, 1. macrourus. The enzyme seems to be inherited in a normal autosomal codominant manner. The gene frequencies for the MDH-A and MDH-A' alleles were O' 82 and 0·18 respectively. The genotypic frequencies indicated that the population was in Hardy-Weinberg equilibrium. None of 22 pouch young from seven litters expressed a phenotype inconsistent with its mother's genotype, assuming autosomal co dominance. Other populations of I. macrourus and of the long-nosed bandicoot (Perameles nasuta) exhibited only the MDH-A allele. Homologous relationships of bandicoot MDH-A2 and MDH-B2 isozymes to those of other vertebrates were established by immunochemical studies.

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“…This column completely removed fraction I protein but had no effect on the MDH activity. Kitto and Kaplan (26) pellet was re-extracted twice more with fresh phosphate buffer. One extraction cycle gave a sample which is representative of the soluble protein of the leaf.…”

Section: Methodsmentioning

confidence: 99%

An Apparent Oligomer of Malate Dehydrogenase from Bean Leaves

Habig

Racusen²

1974

Plant Physiol.

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Purification and Properties of Chicken Heart Mitochondrial and Supernatant Malic Dehydrogenases^*

Cited by 161 publications

References 37 publications

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Genetic Variation and Immunochemical Properties of L-Malate : NAD+ Oxidoreductase Isozymes in the Bandicoot, Isoodon macrourus.

An Apparent Oligomer of Malate Dehydrogenase from Bean Leaves

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Purification and Properties of Chicken Heart Mitochondrial and Supernatant Malic Dehydrogenases*

Cited by 161 publications

References 37 publications

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Genetic Variation and Immunochemical Properties of L-Malate : NAD+ Oxidoreductase Isozymes in the Bandicoot, Isoodon macrourus.

An Apparent Oligomer of Malate Dehydrogenase from Bean Leaves

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Purification and Properties of Chicken Heart Mitochondrial and Supernatant Malic Dehydrogenases^*