Purification to homogeneity of protein kinase C from bovine brain-identity with the phorbol ester receptor.

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Abstract
The calcium‐ and phospholipid‐dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE‐cellulose chromatography, gel filtration and hydrophobic chromatography on octyl‐Sepharose and phenyl‐Sepharose. The phorbol ester receptor co‐purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS‐polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6‐2 and 35‐80 micrograms/ml). These apparent Ka values were reduced 2‐ to 3‐fold by either diolein (20 micrograms/ml) or phorbol‐12,13‐dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol‐12,13‐dibutyrate ([3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine‐dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.