Purification and isotype analysis of protein kinase C from rat liver nuclei

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• 作者：de Moel ; Marië ; lle P. ; Van Emst-De Vries ; Sjenet E. ; Willems ; Peter H.G.M. ; De Pont ; Jan Joep H.H.M.
• 关键词：Nucleus ; Protein kinase C ; ATP ; adenosine triphosphate ; DAG ; diacylglycerol ; DiSBAC₂(3) ; bis-(1 ; 3-diethylthiobarbituric acid)trimethine oxonol ; DTT ; 1 ; 4-dithiothreitol ; EGTA ; ethylene glycol bis(β ; aminoethyl ester) N ; N&prime ; -tetraacetic a
• 刊名：The International Journal of Biochemistry and Cell Biology
• 出版年：1998
• 出版时间：March 27, 1998
• 年：1998
• 卷：30
• 期：2
• 页码：185-195
• 全文大小：767 K

文摘

The properties and subtype composition of protein kinase C present in rat liver nuclei were studied in a Triton-X-100 extract of isolated purified nuclei. The enzyme activity was dependent on both Ca²⁺ and phosphatidylserine, but the phorbol ester 12-O-tetradecanoylphorbol 13-acetate gave only a partial stimulation. Both histone and myelin basic protein served as substrate. Purification of the Triton-X-100 extract followed by Q-Sepharose chromatography gave a preparation with a specific activity of 70 pmol/mg protein min. Western blotting of this preparation showed only the presence of the δ and ζ subtypes, but not the α-subtype, although the latter was present in rat liver homogenates. The β, γ and ε subtypes were not found in the homogenate nor in the nuclear extract. The specific activity of protein kinase C could be further increased up to 800 pmol/mg protein min after protamine agarose chromatography. Also in this preparation the presence of the δ and ζ subtypes could be established.