title: Rapid microassay for protein kinase C translocation in Swiss 3T3 cells.
abstract: The Ca2+/phosphatidylserine-stimulated protein kinase C (PKC) appears to exist as interconvertible inactive, soluble and active, membrane-bound forms. Changes in the bimodal distribution of PKC induced by diacylglycerol or tumor-promoting phorbol esters have been proposed to regulate the activity of this kinase [Nishizuka, Y. (1984) Nature (London) 308, 693-698]. A rapid microassay for assessment of protein kinase C translocation between cytosol and membranes was developed. This procedure, which relied on the selective digitonin-mediated release of cytoplasmic proteins, eliminated potential homogenization and fractionation artifacts. PKC activity toward histone H1 was determined after limited trypsinolysis, which abolished the Ca2+/phospholipid requirement of the enzyme and prevented interference by inhibitory proteins. Complete translocation of PKC to the membrane fraction and subsequent down-regulation of the kinase in response to 12-O-tetradecanoylphorbol-13-acetate treatment of Swiss 3T3 cells could be demonstrated by this method. Platelet-derived growth factor, insulin-like growth factor 1, vasopressin, and prostaglandin F2 alpha facilitated partial conversions of PKC to the membrane-bound form in quiescent 3T3 cells.
mesh_terms: D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006133:Growth Substances / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D006133:Growth Substances; D011493:Protein Kinase C