Stanley C J, Perham R N
Biochem J. 1980 Oct 1;191(1):147-54. doi: 10.1042/bj1910147.
A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.
本文描述了一种新方法，该方法可从牛心组织中并行纯化丙酮酸脱氢酶和2-氧代戊二酸脱氢酶多酶复合物，而无需预先分离线粒体。通过在用于酶复合物初始提取的缓冲液中加入非离子型去污剂（如 Triton X-100 或吐温 80），可使破碎组织中2-氧代酸脱氢酶复合物的所有可检测活性溶解。丙酮酸脱氢酶和2-氧代戊二酸脱氢酶复合物的产量比以前的方法高出许多倍。就比催化活性、十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上的条带模式、沉降特性以及与丙酮酸脱氢酶复合物结合的调节性磷酸激酶而言，通过新方法制备的2-氧代酸脱氢酶复合物与先前研究人员描述的复合物非常相似。使用 Triton X-100 或吐温 80 作为增溶剂极大地提高了2-氧代酸脱氢酶复合物的产量，这支持了大部分丙酮酸脱氢酶复合物以某种方式与线粒体内膜相关联，而不是游离在线粒体基质空间中的可能性。
