Purification and some properties of extramitochondrial malic enzyme from rat skeletal muscle.
Extramitochondrial malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) has been isolated from postmitochondrial supernatant of rat skeletal muscle, by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, Sepharose 6B, ADP-Sepharose and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis. Specific activity of purified enzyme was 20 mumol . min-1 per mg protein, which corresponds to about 3000-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be 264 000. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis showed one polypeptide band of molecular weight 63 000. Thus, it appears that the native protein is a tetramer composed of identical molecular weight subunits. The isoelectric point of the isolated enzyme was at pH 6.15. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of bicarbonate and pyruvate at about 80% of the rate of the forward reaction. The Km values, determined at pH 7.2 for malate and NADP, were 0.125 mM and 11 microM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 4.0 mM, 6.6 microM and 24 mM, respectively. The optimum pH for carboxylation reaction was at pH 7.1. The optimum pH for decarboxylation reaction varied with the malate concentration. The purified malic enzyme catalyzed the decarboxylation of oxaloacetate at pH 4.5. In a system consisting of isolated rat skeletal muscle mitochondria, pyruvate, bicarbonate and NADPH, cytoplasmic malic enzyme is able to replace added malate in stimulating oxidation of acetyl-CoA formed by oxidative decarboxylation of pyruvate. It is suggested that extramitochondrial malic enzyme might be one of the enzymes involved in the anaplerotic supply of Krebs cycle intermediates in skeletal muscle.
D000818:Animals; D002458:Cell Fractionation; D002845:Chromatography; D003593:Cytoplasm / Q000201:enzymology; D003653:Decarboxylation; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D008293:Malates; D008297:Male; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011773:Pyruvates; D051381:Rats
D008293:Malates; D011773:Pyruvates; D008291:Malate Dehydrogenase