Shimomura Y, Nanaumi N, Suzuki M, Popov K M, Harris R A
Laboratory of Biochemistry of Exercise and Nutrition, University of Tsukuba, Ibaraki, Japan.
Arch Biochem Biophys. 1990 Dec;283(2):293-9. doi: 10.1016/0003-9861(90)90645-f.
Branched-chain alpha-ketoacid dehydrogenase kinase was purified to homogeneity from rat liver and rat heart. The initial step was the purification of rat liver and heart branched-chain alpha-ketoacid dehydrogenase complex with high kinase activity by a modification of a method described previously. Preservation of high kinase activity during purification of the complex required the presence of fresh dithiothreitol throughout the procedure. The kinase was released from the complex by oxidation of dithiothreitol with potassium ferricyanide and purified by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography. Both kinase preparations gave only one polypeptide band with a molecular weight of 44,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex by the purified kinase was inhibited by alpha-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex. The kinase did not exhibit autophosphorylation and does not correspond to the same protein as pyruvate dehydrogenase kinase. The kinase phosphorylated histone (type II-S), but this reaction was slow relative to the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex and was not inhibited by alpha-chloroisocaproate.
支链α-酮酸脱氢酶激酶从大鼠肝脏和大鼠心脏中纯化至均一状态。第一步是通过对先前所述方法进行改进，纯化具有高激酶活性的大鼠肝脏和心脏支链α-酮酸脱氢酶复合体。在复合体纯化过程中保持高激酶活性需要在整个过程中存在新鲜的二硫苏糖醇。通过用铁氰化钾氧化二硫苏糖醇从复合体中释放激酶，并通过高速离心、免疫吸附色谱和DEAE-葡聚糖凝胶色谱进行纯化。在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳时，两种激酶制剂均仅给出一条分子量为44,000的多肽带。纯化的激酶对支链α-酮酸脱氢酶复合体的磷酸化和失活受到α-氯异己酸和二氯乙酸的抑制，这两种物质是支链α-酮酸脱氢酶复合体磷酸化的既定抑制剂。该激酶未表现出自磷酸化，且与丙酮酸脱氢酶激酶不是同一种蛋白质。该激酶可使组蛋白（II-S型）磷酸化，但相对于支链α-酮酸脱氢酶复合体的磷酸化而言，此反应较慢，且不受α-氯异己酸的抑制。
