PURIFICATION OF STABLE PROTEIN-KINASE-C FROM MOUSE-BRAIN CYTOSOL BY SPECIFIC LIGAND ELUTION USING FAST PROTEIN LIQUID-CHROMATOGRAPHY

  • 1 April 1986
    • journal article
    • research article
    • Vol. 46  (4) , 1966-1971
Abstract
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.degree. 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.

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