Purification and partial characterization of branched-chain alpha-ketoacid dehydrogenase kinase from rat liver and rat heart.
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.
Shimomura
Y
Y
Laboratory of Biochemistry of Exercise and Nutrition, University of Tsukuba, Ibaraki, Japan.
Nanaumi
N
N
Suzuki
M
M
Popov
K M
KM
Harris
R A
RA
D000818:Animals; D000906:Antibodies; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D006882:Hydroxyapatites; D017886:Durapatite; D011494:Protein Kinases; C035627:(3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase