粗糙脉孢菌线粒体2-酮戊二酸脱氢酶和丙酮酸脱氢酶复合物的纯化

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria.

作者信息

Mareck A, Bessam H, Delattre P, Foucher B

出版信息

Biochimie. 1986 Oct-Nov;68(10-11):1175-80. doi: 10.1016/s0300-9084(86)80061-x.

Abstract

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.

摘要

本文描述了一种简单的粗糙脉孢菌线粒体2-氧代戊二酸脱氢酶和丙酮酸脱氢酶复合物的纯化程序。经聚乙二醇分级沉淀、去除巯基蛋白和凝胶过滤色谱后，所得制剂含有两种活性。在硫醇活化的琼脂糖CL-4B上进行共价色谱，在2-氧代戊二酸存在下，2-氧代戊二酸脱氢酶复合物活性可特异性结合，而丙酮酸脱氢酶复合物活性在丙酮酸存在下得以保留。纯化的2-氧代戊二酸脱氢酶复合物在解离条件下电泳显示4条蛋白带，表观分子量分别为160,000、56,200、55,600、52,600，对2-氧代戊二酸的Km值为3.8×10(-4)M。纯化的丙酮酸脱氢酶复合物显示5条蛋白带，表观分子量分别为160,000、57,600、55,600、52,500和37,100，对丙酮酸的Km值为3.2×10(-4)M。