Rat heart branched chain α-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 (α) and 45,000 Da (β) 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 α 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 catatlytic activity of PDH kinase resides in the α subunit. The β 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 order f1 > f2B ≫ f2A ≫ 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.
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