A rapid preparation of human platelet calcium-activated phospholipid-dependent protein kinase.
A rapid, high yield and relatively stable preparation of human platelet calcium-activated phospholipid-dependent protein kinase (protein kinase C) is described. The method is based on the calcium-dependent and reversible binding of the enzyme to platelet membranes and its success is largely due to the immediate separation of the enzyme from the soluble calcium-dependent protease as well as the platelet endogenous substrate in the first step. Different additions to the platelet lysis medium are examined and the most critical are leupeptin as an inhibitor of the protease and mercapto-ethanol to maintain enzyme activity. This protein kinase C preparation can be done in less than 24 hours and yields an enzyme which is about 90% pure with a 15-fold activation by calcium, diolein and phospholipid, and is devoid of any cAMP-dependent protein kinase or myosin light chain kinase activities.
D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008722:Methods; D008970:Molecular Weight; D011493:Protein Kinase C / Q000097:blood* / Q000302:isolation & purification; D013997:Time Factors
D011493:Protein Kinase C; D002118:Calcium