Patel T B, Olson M S
Biochemistry. 1982 Aug 31;21(18):4259-65. doi: 10.1021/bi00261a012.
The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.
在大鼠肝脏线粒体中研究了共价修饰对支链α-酮酸脱氢酶复合体的调节作用。线粒体内钙和镁的耗竭导致支链α-酮酸脱氢酶复合体失活。支链复合体失活后，单独向线粒体孵育体系中添加钙或镁离子只能使酶复合体部分重新激活。然而，同时添加钙和镁能迅速且几乎完全激活支链酶复合体。在线粒体孵育体系中，在已知能激活或失活支链α-酮酸脱氢酶复合体的条件下加入[32P]磷酸盐。本文提供的证据表明，[32P]磷酸盐掺入到线粒体孵育体系十二烷基硫酸钠-聚丙烯酰胺凝胶中分离出的两条主要蛋白带中。标记的线粒体蛋白带在凝胶系统中的迁移与纯化的心脏来源的丙酮酸脱氢酶（脱羧酶，E1）的α亚基以及纯化的肾脏来源的支链α-酮酸脱氢酶（脱羧酶，E1）的α亚基的迁移完全一致。此外，当支链复合体的测量活性降至最低时，掺入支链酶α链中的[32P]磷酸盐量最大。相反，能使酶复合体最大程度激活的孵育条件会使[32P]磷酸盐掺入支链脱氢酶α亚基的量降至最低。
