Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates.
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 approximately 5 x 10(-7) M Ca2+ in the presence of L-alpha-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 microM. 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.
DeVries
G
G
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alta., Canada.
Fraser
E D
ED
Walsh
M P
MP
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002645:Chickens; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000201:enzymology*; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D007477:Ions; D008970:Molecular Weight; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis* / Q000302:isolation & purification; D014357:Trypsin / Q000378:metabolism
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007477:Ions; C015482:protein kinase modulator; D011493:Protein Kinase C; D014357:Trypsin