Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates

Abstract

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+-dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61 500 by gel filtration and 80 000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at ~5 × 10−7 M Ca2+ in the presence of L-α-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2− substrate, for activity. The Km for ATP was determined to be 20 μM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.Key words: protein kinase C, gizzard, inhibitor, endogenous substrates.