Isolation, properties and role in progesterone biosynthesis of cytosolic malic enzyme from human term placenta.

Abstract Cytosolic malic enzyme ( l -malate: NADP oxidoreductase decarboxylating, EC 1.1.1.40) has been isolated and purified from postmitochondrial supernatant 0f human term placenta by ammonium sulphate fractionation, chromatography on diethylaminoethyl- (DEAE-)cellulose, Sepharose 6B, ADP-Sepharose 4B and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis (PAGE). The specific activity of the purified enzyme was 24.0 αmol. min -1 . mg -1 protein, which corresponds t0 about 7500-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be about 250 000. Sodium dodecyl sulphate- (SDS-)PAGE showed one polypeptide band of molecular weight 63 000. It appears that the native protein is a tetramer composed 0f sub its of identical molecular weight. The isoelectric point of the purified malic enzyme was pH 5.55. The enzyme was shown t0 carboxylate pyruvate in the presence 0f high concentrations 0f pyruvate and bicarbonate at about 8o per cent of the rate of the forward reaction. The optimum pH for the carboxylation reaction was pH 7.3, and that for the decarboxylation reaction varied with malate concentration. The Km values, determined at pH 7.2, for malate, NADP + , Mn 2+ , and Mg 2+ were 81 μ M , 10 μ M , 2.5 μ M and 0.6 m M , respectively. The K m values for pyruvate, NADPH and bicarbonate were 4 m M , 25 μ M and 20 m M , respectively. The enzyme converted malate to pyruvate (at pH 6.3) in the presence of 5 m M NAD + at approximately 8o per cent of the maximum rate with NADP + . It exhibited oxaloacetate decarboxylase activity at about 10 per cent of the rate of oxidative decarboxylation of malate (with NADP + as coenzyme) and pyruvate reductase activity at about 4 per cent of the rate of oxidative decarboxylation of malate with NADP + . The oxidative decarboxylation of malate was inhibited by malate at lower values of pH of incubation medium. This inhibition gradually decreased as the pH of the incubation medium increased. No inhibition was observed at pH 8.2. The addition of purified cytoplasmic malic enzyme, pyruvate, bicarbonate and NADPH generating system (consisting of NADP + , glucose 6-phosphate, glucose 6-phosphate dehydrogenase) stimulated about twof old progesterone biosynthesis by the isolated human placental mitochondria. This stimulation was abolished by arsenite and fluorocitrate. A possible role for the cytosolic malic enzyme in the regulation of progesterone biosynthesis in human placenta is discussed.