Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor.
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.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins; D002273:Carcinogens / Q000378:metabolism; D002352:Carrier Proteins; D002417:Cattle; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D011493:Protein Kinase C; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D011955:Receptors, Drug
D029742:Caenorhabditis elegans Proteins; D002273:Carcinogens; D002352:Carrier Proteins; D010703:Phorbol Esters; D011956:Receptors, Cell Surface; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011494:Protein Kinases; D011493:Protein Kinase C