Swierczynski J, Zelewski M, Zołnierowicz S, Klimek J, Marszałek J, Zelewski L
Placenta. 1987 Mar-Apr;8(2):175-84. doi: 10.1016/0143-4004(87)90020-8.
Cytosolic malic enzyme (L-malate: NADP oxidoreductase decarboxylating, EC I. I. I.40) has been isolated and purified from postmitochondrial supernatant of 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 mumol X min-1 X mg-1 protein, which corresponds to 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 of subunits of identical molecular weight. The isoelectric point of the purified malic enzyme was pH 5.55. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of pyruvate and bicarbonate at about 80 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+, Mn2+, and Mg2+ were 81 microM, 10 microM, 2.5 microM and 0.6 mM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 4 mM, 25 microM and 20 mM, respectively. The enzyme converted malate to pyruvate (at pH 6.3) in the presence of 5 mM NAD+ at approximately 80 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 twofold 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.
已通过硫酸铵分级分离、二乙氨基乙基（DEAE）-纤维素柱层析、琼脂糖凝胶6B柱层析、ADP-琼脂糖凝胶4B柱层析以及Ultrogel AcA-34柱层析，从足月人胎盘的线粒体后上清液中分离并纯化出胞质苹果酸酶（L-苹果酸：NADP氧化还原酶脱羧，EC 1.1.1.40），根据聚丙烯酰胺凝胶电泳（PAGE）判断，该酶已达到表观均一。纯化后酶的比活性为24.0 μmol·min⁻¹·mg⁻¹蛋白，这相当于约7500倍的纯化倍数。通过凝胶过滤法测定天然酶的分子量约为250,000。十二烷基硫酸钠（SDS）-PAGE显示出一条分子量为63,000的多肽带。看来天然蛋白是由分子量相同的亚基组成的四聚体。纯化的苹果酸酶的等电点为pH 5.55。在高浓度丙酮酸和碳酸氢盐存在的情况下，该酶能使丙酮酸羧化，羧化反应速率约为正向反应速率的80%。羧化反应的最适pH为7.3，脱羧反应的最适pH随苹果酸浓度而变化。在pH 7.2条件下测定的苹果酸、NADP⁺、Mn²⁺和Mg²⁺的Km值分别为81 μM、10 μM、2.5 μM和0.6 mM。丙酮酸、NADPH和碳酸氢盐的Km值分别为4 mM、25 μM和20 mM。在5 mM NAD⁺存在的情况下（pH 6.3），该酶将苹果酸转化为丙酮酸的速率约为以NADP⁺为底物时最大速率的80%。它表现出草酰乙酸脱羧酶活性，其活性约为苹果酸氧化脱羧（以NADP⁺为辅酶）速率的10%，丙酮酸还原酶活性约为苹果酸以NADP⁺为辅酶氧化脱羧速率的4%。在较低的孵育介质pH值下，苹果酸对苹果酸的氧化脱羧有抑制作用。随着孵育介质pH值的升高，这种抑制作用逐渐减弱。在pH 8.2时未观察到抑制作用。添加纯化的细胞质苹果酸酶、丙酮酸、碳酸氢盐和NADPH生成系统（由NADP⁺、6-磷酸葡萄糖、6-磷酸葡萄糖脱氢酶组成）可使分离的人胎盘线粒体的孕酮生物合成增加约两倍。亚砷酸盐和氟柠檬酸可消除这种刺激作用。本文讨论了胞质苹果酸酶在人胎盘孕酮生物合成调节中的可能作用。
