Characterization and partial purification of rat submandibular gland protein kinase C.
Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 microM and 5 micrograms, respectively. Addition of 10(-6) M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 microM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.
Pon
D J
DJ
Department of Pharmacology, University of Toronto, Ont., Canada.
Lintlop
S P
SP
Sen
A K
AK
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D011493:Protein Kinase C / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013363:Submandibular Gland / Q000201:enzymology*
D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase