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Purification and properties of pyruvate dehydrogenase kinase from bovine kidney.

1983; Elsevier BV; Volume: 258; Issue: 15 Linguagem: Inglês

10.1016/s0021-9258(17)44689-8

1083-351X

Larry R. Stepp, Flora H. Pettit, Stephen J. Yeaman, Lester J. Reed,

Metabolism and Genetic Disorders

Pyruvate dehydrogenase kinase was purified about 2,700-fold to apparent homogeneity from extracts of bovine kidney mitochondria.The kinase consists of two subunits (aB) with molecular weights of 48,000 (a) and 45,000 (B) as estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis.Kinase activity resides in the a subunit.The a subunit is sensitive to proteolysis by chymotrypsin, whereas the , 8 subunit is selectively modified by trypsin.These observations, together with the results of peptide mapping, indicate that the two subunits are distinctly different proteins.It is proposed that the B subunit is a regulatory subunit.The mammalian pyruvate dehydrogenase complex is composed of multiple copies of three major components, pyruvate dehydrogenase, dihydrolipoamide acetyltransferase, and dihydrolipoamide dehydrogenase.The dihydrolipoamide acetyltransferase component forms a structural core, composed of 60 subunits arranged with icosahedral 532 symmetry, to which pyruvate dehydrogenase and dihydrolipoamide dehydrogenase are bound by noncovalent bonds (for a review, see Ref. 1).The mammalian complex also contains small amounts of two regulatory enzymes, a kinase and a phosphatase, that modulate the activity of pyruvate dehydrogenase by phosphorylation (inactivation) and dephosphorylation (activation), respectively.Pyruvate dehydrogenase kinase is tightly bound to dihydrolipoamide acetyltransferase and copurifies with the complex, whereas pyruvate dehydrogenase phosphatase is loosely associated with the complex (2, 3).In this paper, we report the purification to apparent homogeneity and some properties of pyruvate dehydrogenase kinase from bovine kidney mitochondria. EXPERIMENTAL PROCEDURESMaterials-Highly purified pyruvate dehydrogenase complex and crystalline pyruvate dehydrogenase from bovine kidney mitochondria were prepared as described previously (2, 3 ) .Staphylococcus aureus V8 protease was obtained from Miles Laboratories, L-1-(tosy1amido)-2-phenylethyl chloromethyl ketone-treated trypsin was from Millipore, N-a-p-tosyl-L-lysine chloromethyl ketone-treated a-chymotrypsin (Type VII), thermolysin, soybean trypsin inihibitor, bovine serum albumin, calmodulin, rabbit muscle phosphorylase b and glycogen synthase, and histones 11-A, VI-S, and VIII-S were from Sigma, elastase was from Boehringer Mannheim, and N a T and [y-"PIATP were from Amersham Corp.All other reagents and materials were of