Buxton D B, Olson M S
J Biol Chem. 1982 Dec 25;257(24):15026-9.
Phosphorylation of the alpha-subunits of the branched chain alpha-keto acid and the pyruvate dehydrogenases was measured in mitochondria isolated from rat hearts perfused in the presence of [32P]phosphate and various substrates. Mitochondrial isolations were accomplished rapidly under conditions which would preclude the interconversion of these two enzyme complexes between their active (dephosphorylated) and inactive (phosphorylated) forms. Inactivation of the branched chain complex and the concomitant incorporation of [32P]phosphate into the alpha-chain of the dehydrogenase subcomponent were observed in hearts perfused with glucose (10 mM) or pyruvate (10 mM). alpha-Ketoisocaproate infusion caused a 15-fold activation and dephosphorylation of the branched chain dehydrogenase, while pyruvate dehydrogenase remained inactive and phosphorylated under these conditions. Both dehydrogenase components were partially activated, and most of the [32P]phosphate was removed from their respective alpha-subunits during substrate-free perfusion. Both enzyme complexes were activated and dephosphorylated completely during infusion of dichloroacetate (1 mM) and glucose (10 mM).
在存在[32P]磷酸盐和各种底物的情况下，对灌注的大鼠心脏分离出的线粒体中支链α-酮酸脱氢酶和丙酮酸脱氢酶的α亚基磷酸化进行了测定。线粒体分离在能够防止这两种酶复合物在其活性（去磷酸化）和无活性（磷酸化）形式之间相互转化的条件下快速完成。在用葡萄糖（10 mM）或丙酮酸（10 mM）灌注的心脏中，观察到支链复合物失活以及[32P]磷酸盐伴随掺入脱氢酶亚组分的α链中。输注α-酮异己酸导致支链脱氢酶激活15倍并去磷酸化，而丙酮酸脱氢酶在此条件下仍保持无活性且磷酸化。在无底物灌注期间，两种脱氢酶组分均被部分激活，并且大部分[32P]磷酸盐从其各自的α亚基中去除。在输注二氯乙酸（1 mM）和葡萄糖（10 mM）期间，两种酶复合物均被完全激活并去磷酸化。
