Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.
A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.
Dell
K R
KR
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Canada.
Walsh
M P
MP
Severson
D L
DL
D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006657:Histones / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D017830:Octoxynol; D010718:Phosphatidylserines / Q000494:pharmacology; D010750:Phosphoproteins; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001798:Blood Proteins; D004075:Diglycerides; D006657:Histones; D010718:Phosphatidylserines; D010750:Phosphoproteins; C039077:platelet protein P47; D011092:Polyethylene Glycols; D017830:Octoxynol; D011493:Protein Kinase C; D002118:Calcium; C013965:diolein