A three-step purification procedure for protein kinase C: characterization of the purified enzyme.
A three-step purification procedure for protein kinase C: characterization of the purified enzyme.
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蛋白激酶 C 的三步纯化程序：纯化酶的表征。
DOI：
10.1016/0003-2697(87)90471-4
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发表时间：
1987
影响因子：
2.9
通讯作者：
Gill,GN
中科院分区：
文献类型：
--
作者：
Walton,GM;Bertics,PJ;Hudson,LG;Vedvick,TS;Gill,GN
本文介绍了一种由离子交换层析、疏水层析和底物亲和层析组成的蛋白激酶C的高效、高产三步纯化方法。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上表现出均一性的蛋白质含有根据cDNA克隆预测的牛脑酶α和β同工酶形式的氨基酸序列。这两种形式都表现出活性，如由[3 H]佛波醇二丁酸酯结合化学计量学所指示的。纯化的酶是活性作为一个单体物种，表现出高协同性之间的Ca+2和磷脂酰丝氨酸结合的活性，并经历分子内的自我磷酸化在丝氨酸和苏氨酸残基。酶与ATP一起孵育，导致广泛的自我磷酸化，显着稳定磷酸转移酶活性，而不会增加反应的Vmax。
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 α and β isoenzyme forms of the bovine brain enzyme. Both forms appear active as indicated by [3H]phorbol dibutyrate binding stoichiometry of ∼ 1. Purified enzyme is active as a monomeric species, exhibits high cooperativity between Ca+2and 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 Vmaxof the reaction.