title: A three-step purification procedure for protein kinase C: characterization of the purified enzyme.
abstract: An efficient high yield three-step purification procedure for protein kinase C consisting of ion exchange, hydrophobic interaction, and substrate affinity chromatographies is described. Protein which appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis contains amino acid sequences, predicted from cDNA cloning, for both the alpha and beta isoenzyme forms of the bovine brain enzyme. Both forms appear active as indicated by [3H]phorbol dibutyrate binding stoichiometry of approximately 1. Purified enzyme is active as a monomeric species, exhibits high cooperativity between Ca+2 and phosphatidylserine binding for activity, and undergoes intramolecular self-phosphorylation at both serine and threonine residues. Incubation of the enzyme with ATP, which leads to extensive self-phosphorylation, markedly stabilizes phosphotransferase activity without increasing the Vmax of the reaction.
authors.forename: G M
authors.initials: GM
authors.lastname: Walton
authors.forename: P J
authors.initials: PJ
authors.lastname: Bertics
authors.forename: L G
authors.initials: LG
authors.lastname: Hudson
authors.forename: T S
authors.initials: TS
authors.lastname: Vedvick
authors.forename: G N
authors.initials: GN
authors.lastname: Gill
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010703:Phorbol Esters; D015240:Phorbol 12,13-Dibutyrate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C