Determination of Ca2+- and phospholipid-dependent protein kinase in rat liver membranes.
A method has been developed to measure the Ca2+- and phospholipid-dependent protein kinase in membrane fractions. The method is based on the fact that this enzyme is resistant to comparatively high concentrations of octylglycoside. Rat liver membranes were treated with octylglycoside and the phosphate incorporation from [gamma-32P]ATP was measured in the presence of histone H1. The enzyme activity was determined as the difference between the incorporation obtained after addition of Ca2+ and phosphatidylserine and the incorporation obtained without these additions but with EGTA. The endogenous incorporation of phosphate to membrane components was constant under these incubation conditions. The conditions for determination of the membrane-bound enzyme were optimized. Two thirds of the total enzymic activity was attached to membranes in rat liver cells. A highly purified plasma membrane preparation had the highest specific activity, while most of the bound enzyme was found in microsomes, and only traces were found in mitochondria.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004533:Egtazic Acid / Q000494:pharmacology; D005960:Glucosides; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D003902:Detergents; D005960:Glucosides; D006657:Histones; D010718:Phosphatidylserines; C018619:octyl-beta-D-glucoside; D004533:Egtazic Acid; D011494:Protein Kinases; D002118:Calcium