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

Abstract

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 Ca 2+ 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.