Le Deaut J Y, Ledig M, Mandel P
Biochimie. 1976 Nov 13;58(9):1017-29. doi: 10.1016/s0300-9084(76)80081-8.
A method for isolation of a soluble ATPase from rat liver mitochondria after freeze thaw cycling is described. Two enzymatically active fractions were separated by DEAE-cellulose chromatography (ATPase 1 and ATPase 2). ATPase 1 has been purified 300 fold. ATPase 1 was homogenous as judged by polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8-6.0 and the optimum temperature was 45 degrees C. The enzyme follows Michaelis-Menten kinetics: Km (9 X 10(-4) M), Vmax (23,6 mumoles Pi released X min -1 X mg protein -1). The enzyme hydrolysed nucleoside triphosphates, but was inactive upon nucleoside di and monophosphates, glucose 6-phosphate, phosphoserine, pyrophosphate and glycerol 2-phosphate. In contrast to membrane bound ATPase, cations have no effect on the enzyme activity. Nucleoside di and mono phosphates and glycerol 2 phosphate inhibited competitively the enzyme. The enzyme was not affected by oligomycin, but was stimulated by lactate, 2-mercaptoethanol and dithiothreitol.
本文描述了一种在冻融循环后从大鼠肝脏线粒体中分离可溶性ATP酶的方法。通过DEAE-纤维素色谱法分离出两个具有酶活性的组分（ATP酶1和ATP酶2）。ATP酶1已被纯化300倍。根据聚丙烯酰胺凝胶电泳判断，ATP酶1是均一的。该酶的最适pH为5.8 - 6.0，最适温度为45℃。该酶遵循米氏动力学：Km（9×10⁻⁴M），Vmax（23.6微摩尔无机磷释放量×分钟⁻¹×毫克蛋白⁻¹）。该酶能水解核苷三磷酸，但对核苷二磷酸和单磷酸、6-磷酸葡萄糖、磷酸丝氨酸、焦磷酸和2-磷酸甘油无活性。与膜结合ATP酶不同，阳离子对该酶活性无影响。核苷二磷酸和单磷酸以及2-磷酸甘油竞争性抑制该酶。该酶不受寡霉素影响，但受乳酸、2-巯基乙醇和二硫苏糖醇刺激。
