Rapid purification of protein kinase C by high performance liquid chromatography☆

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Abstract

Protein kinase C was purified from rat brain cytosol by using a high performance liquid chromatography (HPLC), Pharmacia FPLC system. This procedure employed a column chromatography on DE-52, followed by three steps of HPLC procedures with threonine-Sepharose (prepared as described in this report), TSK gel Phenyl-5PW (Toyo Soda), and TSK gel G3000SW (Toyo Soda) columns. Starting from about 30 g of rat brain, approximately 200 μg of pure enzyme was obtained. The procedure was very simple and highly reproducible. The enzyme thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 10% (wv) glycerol and 0.05% (wv) Triton X-100, the enzyme could be stored at −80°C for several months.

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This investigation has been supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture (1984–1985), the Science and Technology Agency (1984–1985), the Yamanouchi Foundation for Research on Metabolic Disorders (1984–1985), the Muscular Dystrophy Association (1985–1986), the Foundation for the Promotion of Research on Medical Resources (1985), Merck Sharp & Dohm Research Laboratories (1985), Biotechnology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd. (1985), and Central Research Laboratories, Ajinomoto Co. Ltd. (1985).

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