PURIFICATION OF 2-OXO ACID DEHYDROGENASE MULTI-ENZYME COMPLEXES FROM OX HEART BY A NEW METHOD
PURIFICATION OF 2-OXO ACID DEHYDROGENASE MULTI-ENZYME COMPLEXES FROM OX HEART BY A NEW METHOD
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DOI：
10.1042/bj1910147
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发表时间：
1980-01-01
影响因子：
4.1
通讯作者：
PERHAM, RN
中科院分区：
文献类型：
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作者：
STANLEY, CJ;PERHAM, RN
本文介绍了一种新的方法，该方法允许从牛心脏中平行纯化丙酮酸脱氢酶和2-酮戊二酸脱氢酶多酶复合物，而不需要预先分离线粒体。通过在用于酶复合物的初始提取的缓冲液中包含非离子去污剂如Triton X-100或Tween-80，使破裂组织中2-含氧酸脱氢酶复合物的所有可测定活性可溶。丙酮酸脱氢酶和2-氧戊二酸脱氢酶复合物的产率比通过以前的方法获得的产率高出许多倍。在特定的催化活性，十二烷基硫酸钠/聚丙烯酰胺凝胶电泳，沉降性能和拥有的调节磷酸激酶结合的丙酮酸脱氢酶复合物的带型，2-含氧酸脱氢酶复合物的制备的新方法非常类似于以前的工人所描述的。通过使用Triton X-100或Tween-80作为增溶剂，大大提高了2-含氧酸脱氢酶复合物的产率，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结合而不是游离在线粒体基质空间中的可能性。
A new method is described that allows the parallel purification of the pyruvate dehydrogense 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 sulfate/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 dehydrogense complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.