Taroni F, Gellera C, Di Donato S
Laboratory of Biochemistry, Istituto Neurologico C. Besta, Milan, Italy.
Biochim Biophys Acta. 1987 Dec 18;916(3):446-54. doi: 10.1016/0167-4838(87)90191-9.
Human muscle mitochondria reduced either NADP+ or NAD+ in the presence of L-malate and Mn2+ or Mg2+. After polyacrylamide slab gel electrophoresis and agarose gel isoelectrofocusing, two bands were seen in mitochondrial extract, one strictly NADP+-dependent and the other reacting with either NAD+ or NADP+. The two mitochondrial malic enzymes were separated by DEAE-Sepharose chromatography. The NAD+/NADP+-dependent enzyme was purified 1600-fold with a final yield of 34% and a final specific activity of 32.9 units/mg of protein by employing affinity chromatography on Agarose-ATP. SDS electrophoresis revealed a single band having an apparent Mr = 64,000. Estimates of the native apparent molecular weight upon gel filtration yielded a value of 140,300. Kinetic characterization showed that succinate and ATP were activator and inhibitor, respectively. In the absence of succinate the Km values for malate, NAD+ and NADP+ were 3.7, 0.13 and 0.78 mM, respectively; in the presence of succinate the Km value for malate was 1.9 mM. ATP was found to be an inhibitor competitive with malate, with a Ki (ATP) of 0.2 mM. This is the first report to show that mammalian skeletal muscle mitochondria contains two distinct malic enzymes, one active with either NAD+ or NADP+ and the other active only with NADP+.
在L-苹果酸以及锰离子或镁离子存在的情况下，人类肌肉线粒体可使NADP⁺或NAD⁺还原。经过聚丙烯酰胺平板凝胶电泳和琼脂糖凝胶等电聚焦后，在线粒体提取物中可见两条带，一条严格依赖NADP⁺，另一条则与NAD⁺或NADP⁺都能发生反应。通过DEAE-琼脂糖凝胶柱层析分离出了两种线粒体苹果酸酶。利用琼脂糖-ATP亲和层析法，将依赖NAD⁺/NADP⁺的酶纯化了1600倍，最终产率为34%，最终比活性为32.9单位/毫克蛋白质。SDS电泳显示有一条表观分子量Mr = 64,000的单带。凝胶过滤法测得的天然表观分子量为140,300。动力学特性表明，琥珀酸和ATP分别是激活剂和抑制剂。在没有琥珀酸的情况下，苹果酸、NAD⁺和NADP⁺的Km值分别为3.7、0.13和0.78 mM；在有琥珀酸的情况下，苹果酸的Km值为1.9 mM。发现ATP是与苹果酸竞争的抑制剂，其Ki（ATP）为0.2 mM。这是首次报道哺乳动物骨骼肌线粒体含有两种不同的苹果酸酶，一种对NAD⁺或NADP⁺都有活性，另一种仅对NADP⁺有活性。
