Swierczyński J
Biochim Biophys Acta. 1980 Nov 6;616(1):10-21. doi: 10.1016/0005-2744(80)90258-2.
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.
已从大鼠骨骼肌的线粒体后上清液中分离出胞质苹果酸酶（L-苹果酸：NADP⁺氧化还原酶（草酰乙酸脱羧），EC 1.1.1.40），通过硫酸铵分级分离、DEAE-纤维素柱层析、琼脂糖凝胶6B柱层析、ADP-琼脂糖柱层析和Ultrogel AcA-34柱层析，直至聚丙烯酰胺凝胶电泳显示达到表观均一性。纯化酶的比活性为每毫克蛋白质20 μmol·min⁻¹，这相当于约3000倍的纯化倍数。通过凝胶过滤测定天然酶的分子量为264000。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳显示一条分子量为63000的多肽带。因此，天然蛋白质似乎是由相同分子量亚基组成的四聚体。分离出的酶的等电点为pH 6.15。在高浓度碳酸氢盐和丙酮酸存在下，该酶使丙酮酸羧化的速率约为正向反应速率的80%。在pH 7.2下测定的苹果酸和NADP的Km值分别为0.125 mM和11 μM。丙酮酸、NADPH和碳酸氢盐的Km值分别为4.0 mM、6.6 μM和24 mM。羧化反应的最适pH为7.1。脱羧反应的最适pH随苹果酸浓度而变化。纯化的苹果酸酶在pH 4.5时催化草酰乙酸的脱羧反应。在由分离的大鼠骨骼肌线粒体、丙酮酸、碳酸氢盐和NADPH组成的体系中，胞质苹果酸酶能够替代添加的苹果酸，刺激丙酮酸氧化脱羧形成的乙酰辅酶A的氧化。提示胞质苹果酸酶可能是参与骨骼肌中三羧酸循环中间产物回补供应的酶之一。
