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Purification of Stable Protein Kinase C from Mouse Brain Cytosol by Specific Ligand Elution Using Fast Protein Liquid Chromatography

Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, NIH, Bethesda, Maryland 20892

The Ca²⁺- and phospholipid-dependent protein kinase (protein kinase C) has been purified to electrophoretic homogeneity from mouse brain cytosol. [20-³H]phorbol 12,13-dibutyrate binding activity was found to coelute quantitatively with the protein kinase activity throughout the purification procedure. The crude extract was first run over a DE52 column. Fractions containing peak activities were then chromatographed using a fast protein liquid chromatography system with a Mono Q column followed by chromatography on the same column run in the presence of 1 mM adenosine triphosphate. The adenosine triphosphate specifically shifted the elution position of the Ca²⁺- and phospholipid-dependent protein kinase, providing a powerful step in the purification procedure. The remaining minor contaminants were removed by hydrophobic chromatography on a TSK-phenyl-5-PW column. This purification procedure required less than 2 days after the initial large batch DE52 column chromatography. The molecular weight of the purified receptor was estimated to be 81,000 by its mobility on sodium dodecyl sulfate/polyacrylamide gels, in agreement with the published values. Optimal conditions for the storage of the purified receptor were sought. Both protein kinase and phorbol ester binding activities were stable for 2 mo when stored in the presence of 0.01% Triton X-100 at -70°C. Polyclonal antibodies to the purified receptor have been prepared from rabbits. These antibodies recognized the purified receptor in electroblotting assays and were able to immunoprecipitate the purified receptor.

¹ Present address: Neuroscience/Cardiovascular Section, CIBA-GEIGY Corp., 556 Morris Avenue, Summit, NJ 07901. To whom requests for reprints should be addressed.

Received 7/30/85. Revised 12/ 9/85. Accepted 12/18/85.