Popov K M, Shimomura Y, Harris R A
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Protein Expr Purif. 1991 Aug;2(4):278-86. doi: 10.1016/1046-5928(91)90084-v.
Rat heart branched chain alpha-ketoacid dehydrogenase kinase (BCKDH kinase) and pyruvate dehydrogenase kinase (PDH kinase) were purified from their respective complexes to apparent homogeneity. BCKDH kinase consisted of one subunit with molecular weight 44,000-45,000 Da, whereas PDH kinase consisted of two subunits with molecular weight 48,000 Da (alpha) and 45,000 Da (beta) as previously shown for the bovine kidney enzyme (Stepp et al., 1983, J. Biol. Chem. 258, 9454-9458). Proteolysis maps of BCKDH kinase and the two subunits of PDH kinase were different, suggesting that all subunits are different entities. The alpha subunit of the rat heart PDH kinase could be cleaved selectively by chymotrypsin with concomitant loss of kinase activity, as previously shown for the bovine kidney enzyme, suggesting that the catalytic activity of PDH kinase resides in the alpha subunit. The beta subunit appeared to be a different entity unique to the PDH kinase. Both kinases exhibited marked substrate specificity toward their respective complexes and would not inactivate heterologous complexes. The kinases possessed slightly different substrate specificity toward histones. BCKDH kinase preferentially phosphorylated histones in the order f1 greater than f2B much greater than f2A much greater than f3. The relative order for PDH kinase was the same, but f2A and f3 were considerably better substrates than they were for BCKDH kinase. These observations suggest that the kinases have different requirements for the structure of the protein at their phosphorylation sites.
大鼠心脏支链α-酮酸脱氢酶激酶（BCKDH激酶）和丙酮酸脱氢酶激酶（PDH激酶）从各自的复合物中纯化至表观均一。BCKDH激酶由一个分子量为44,000 - 45,000道尔顿的亚基组成，而PDH激酶由两个亚基组成，分子量分别为48,000道尔顿（α）和45,000道尔顿（β），如先前对牛肾酶的研究所示（Stepp等人，1983年，《生物化学杂志》258卷，9454 - 9458页）。BCKDH激酶和PDH激酶两个亚基的蛋白酶解图谱不同，表明所有亚基都是不同的实体。大鼠心脏PDH激酶的α亚基可被胰凝乳蛋白酶选择性切割，同时激酶活性丧失，如先前对牛肾酶的研究所示，这表明PDH激酶的催化活性存在于α亚基中。β亚基似乎是PDH激酶特有的不同实体。两种激酶对各自的复合物都表现出明显的底物特异性，不会使异源复合物失活。这两种激酶对组蛋白的底物特异性略有不同。BCKDH激酶优先磷酸化组蛋白的顺序为f1＞f2B＞＞f2A＞＞f3。PDH激酶的相对顺序相同，但f2A和f3作为底物比它们对BCKDH激酶时要好得多。这些观察结果表明，激酶对其磷酸化位点处蛋白质的结构有不同的要求。
