Purification and characterization of protein kinase C isozymes from rat heart.
A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca2+ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca2+ and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca2+.
Qu
Y
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Department of Pharmacology, Faculty of Medicine, University of Toronto, Canada.
Torchia
J
J
Phan
T D
TD
Sen
A K
AK
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D004075:Diglycerides; D007527:Isoenzymes; D010718:Phosphatidylserines; D010743:Phospholipids; D011493:Protein Kinase C; D002118:Calcium