Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase.
The catalytic subunit of rabbit skeletal muscle cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been isolated in pure form. It has a molecular weight of 41,300, as determined by sedimentation equilibrium, which is in good agreement with the value of 41,000 determined by electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation velocity determinations indicate that the subunit has an S20,w value of 3.12 which is essentially independent of protein concentration. These experiments are interpreted as indicating that the catalytic subunit dissociated from the holoenzyme exists as a monomer in solution. The least abundant amino acid is half-cystine, which was calculated to be present at 2.8 mol/mol of protein. The sulfhydryl reagents, N-ethylmaleimide, p-chloromercuribenzoic acid, and 5,5'-dithiobis(2-nitrobenzoic acid) inhibit the enzymatic activity of the subunit; inhibition by the two latter compounds can be reversed by 2-mercaptoethanol. Binding of 1 mol of N-ethylmaleimide/mol of protein results in almost complete inhibition. The isolated catalytic subunit contains 2.2 mol of tightly bound phosphate/mol of protein. Identification of either O-phosphoserine or O-phosphothreonine after partial acid hydrolysis indicates that at least part of the endogeneous phosphate exists as the phospho ester of one of these amino acids. Two peaks of catalytic activity corresponding to isoelectric points of pH 7.4 and 8.5 were identified by isoelectric focusing. Both forms utilize the same substrates and have similar sedimentation constants.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002729:Chloromercuribenzoates; D046911:Macromolecular Substances; D004228:Dithionitrobenzoic Acid; D000242:Cyclic AMP; D011494:Protein Kinases; D005033:Ethylmaleimide