Isolation and properties of malic dehydrogenase from ox-heart mitochondria

Davies, Daniel; Kun, Ernest

doi:10.1042/bj0660307

Cited by 137 publications

(31 citation statements)

References 17 publications

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“…The pH optima for both MDH and LDH activity in the fraction of the hemolysate corresponding in mobility to the a2-globulin was 8 In this connection it is pertinent that Meister has shown that many different a-keto and a-diketo acids, including oxalacetic acid, can serve as substrates for LDH obtained from rabbit muscle (7). In addition, Davies and Kun have described several a-hydroxy dicarboxylic acids which act as substrates for MDH obtained from pig heart (8). Recently, alcohol dehydrogenase from yeast has been shown to oxidize lactate and reduce pyruvate (9).…”

Section: Resultsmentioning

confidence: 99%

Observations on the Heterogeneity of Malic and Lactic Dehydrogenase in Human Serum and Red Blood Cells1

Vesell¹,

Bearn²

1958

J. Clin. Invest.

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The enzyme malic dehydrogenase (MDH)2 catalyzes the conversion of oxalacetic acid to malic acid. It The method of zone electrophoresis, using a starch supporting medium in barbital buffer, pH 8.6, with an ionic strength of 0.1 or 0.05, was used to separate the serum proteins (4). After electrophoresis the starch block was cut into half inch segments, the protein eluted with 5.0 ml. buffer and its concentration determined by a modification of the Folin-Ciocalteau procedure (4). An aliquot of 2.5 ml. from each eluate was then assayed for MDH activity spectrophotometrically by the method of Wacker, Ulmer, and Vallee (5). Each eluate was incubated for 20 minutes at room temperature with 0.2 ml. of 0.003 molar DPNH. The mixture was transferred to a Beckman cuvette of 1 cm. path length; 0.1 ml. of 0.0076 molar oxalacetate was added, and the decrease in absorption at 340 mju was measured in the Beckman spectrophotometer. Readings were obtained every 30 seconds for 3 minutes. One unit of dehydrogenase activity was defined as a decrease in the optical density at 340 mu of 0.001 per minute. Under these conditions, the contribution to the observed MDH activity by the simultaneous measurement of LDH, caused by spontaneous decarboxylation of oxalacetate to form pyruvate, was found to be negligible. Lactic dehydrogenase obtained from rabbit muscle (Worthington laboratories) was found to have insignificant quantities of activity when oxalacetate was used as substrate instead of pyruvate.In several experiments, a 2.5 ml. sample of an hemolysate prepared from normal human red cells (6) was fractionated electrophoretically and an aliquot of the eluate was assayed for MDH activity. To determine the pH optima of the three peaks, 0.3 ml. was removed from the tube containing the maximum MDH activity, and added to tubes containing barbital buffers varying in pH from 7.4 to 9.2, all at 0.1 ionic strength. MDH activity was then reassayed.The effect of the inhibitors, ethylenediaminetetraacetic acid and p-chloromercuribenzoate, on each of the activity peaks was studied according to the method outlined below. Immediately after the serum fractions had been assayed for enzymatic activity the inhibitor was added, the solution thoroughly mixed and the activity redetermined. The decline in activity between the two measurements was attributed to the action of the inhibitor.The final concentrations of ethylenediaminetetraacetic 672

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Section: Resultsmentioning

confidence: 99%

Observations on the Heterogeneity of Malic and Lactic Dehydrogenase in Human Serum and Red Blood Cells1

Vesell¹,

Bearn²

1958

J. Clin. Invest.

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“…Some slow oxidation of meso and D-tartaric acids was observed (¼ 2.7% of activity against L-malate), which has been noted previously for m-MDH from bovine heart [59,60]. However, unlike the bovine enzyme, the T. emersonii m-MDH displayed no activity with mesoxalate, hydroxymalonate and a-ketoglutarate [60].…”

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

confidence: 99%

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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“…The possibility exists that inside the intact mitochondrion, the enzyme may react to salinity in a different way than the liberated enzyme. It was suggested that the malic dehydrogenase is located in the external membrane of the mitochondrion (7,17); if this is so, permeability should not be a limiting factor. However, respiratory control mechanisms, functioning in the intact particle, may cause a different response to the effect of NaCl and other salts than in free enzymes.…”

Section: Methodsmentioning

confidence: 99%

Malic Dehydrogenase from Tamarix Roots

Kalir¹,

Poljakoff‐Mayber²

1975

Plant Physiol.

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Soluble and mitochondrial malic dehydrogenases (MDH) were isolated from root tips of the halophyte Tamarix tetragyna L. grown in the presence and absence of NaCI. The activity of the enzymes isolated from root tips grown in the presence of NaCI was lower than that of the enzymes isolated from roots grown in absence of NaCl. The mitochondrial MDH was much more sensitive to salinity than the soluble MDH. The soluble enzyme from roots grown in NaCl had a higher Km for malate and lower Km for NAD than enzyme from the control roots.Addition of NaCl in vitro at 72 mM significantly stimulated the reductive activity of soluble MDH, while higher NaCl concentrations (240 mM and above) depressed enzyme activity. Another group of organisms showing a very high tolerance to substrate salinity is the group of the halophilic bacteria. The enzymes of these organisms are apparently adapted to function in the presence of high ionic concentrations and are activated by them (1, 2, 6, 13-16, 21, 22). Holmes and Halvorson (13) studied the effect of NaCl on malic dehydrogenase from obligatory halophilic bacteria, facultative halophilic bacteria, and animal tissue. The enyzme from the animal tissue showed maximal activity in the absence of NaCl and the activity decreased with increasing NaCl concentration. The enzyme isolated from obligatory halophilic bacteria, on the other hand, showed the highest activity at the high NaCl concentrations (up to 4 M) and its activity decreased with decreasing NaCl concentrations. Hubbard and Miller (14,15) showed similar to halophilic bacteria, or whether the enzyme was simiIt was of interest, therefore, to examine whether an enzyme of a halophytic plant showed salt requirement for its activity, similar to halophilic bacteria, or whether the enzyme was similar to that of the glycophytes. Malic dehydrogenase (L-malate-NAD oxidoreductase EC 1 .1 .1 . 37) isolated from Tamarix roots was examined. MATERIALS AND METHODSCuttings of Tamarix tetragyna L. were collected from a tree from Mediterranean coastal salt marshes in the Naaman area and were rooted and grown in vermiculite moistened either with half strength Hoagland's solution (12) or with Hoagland's solution containing 0.12 M NaCl (osmotic potential -v = -5 atm). When the cuttings began to root, salinity was stepped up to final salinities of 0.12 M, 0.24 M, 0.36 M, and sometimes 0.48 M NaCl (corresponding to . = -5, -10, -15, and -20 atm, respectively) and higher. Loss of water was compensated daily.Isolation of Mitochondria and Preparation of Enzyme. Root tips, 2 cm long, were collected and blotted with filter paper. Two g of root were ground with sand and 0.5 g of Polyclar AT in 0.1 M tris-HCI buffer (pH 7.4) containing 0.5 M sucrose, 5 mM EDTA, and 1 mm dithioerythritol (buffer I in Fig. 1) and treated as outlined in Figure 1.For kinetic studies, the soluble and the solubilized enzymes were partially purified by precipitation with solid (NH4)2SO,.

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Isolation and properties of malic dehydrogenase from ox-heart mitochondria

Cited by 137 publications

References 17 publications

Observations on the Heterogeneity of Malic and Lactic Dehydrogenase in Human Serum and Red Blood Cells1

Observations on the Heterogeneity of Malic and Lactic Dehydrogenase in Human Serum and Red Blood Cells1

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Malic Dehydrogenase from Tamarix Roots

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