Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine.
A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids and diacylglycerols. Triton X-100 mixed micelles containing 8 mol % phosphatidylserine (PS) and 2.5 mol % sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1 did in the standard assay. However, protein kinase C activity was more responsive to diC18:1 in the mixed micellar assay than the standard assay. At 8 mol % PS and 100 microM Ca2+, diC18:1 stimulated maximally at 1 mol %. At 2.5 mol % diC18:1 and 100 microM Ca2+, PS did not activate until 3 mol % and then did so cooperatively with maximal stimulation occurring at 6-8 mol %. Direct evidence for a Ca2+-, PS-, and diC18:1-dependent interaction of protein kinase C with mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data permit inferences pertaining to the number of diC18:1 and PS molecules/micelle which are required for activation. For diC18:1, a single molecule may be sufficient but no more than 2 molecules are required. For PS, greater than 4 but less than 10 molecules are required. These data establish that a phospholipid bilayer is not required for protein kinase C activation and that activation of monomeric protein kinase C occurs.
Hannun
Y A
YA
Loomis
C R
CR
Bell
R M
RM
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D004075:Diglycerides / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D005989:Glycerides / Q000494:pharmacology*; D007700:Kinetics; D008823:Micelles; D017830:Octoxynol; D010718:Phosphatidylserines / Q000494:pharmacology*; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011493:Protein Kinase C; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D004075:Diglycerides; D005989:Glycerides; D008823:Micelles; D010718:Phosphatidylserines; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D004533:Egtazic Acid; D017830:Octoxynol; D011494:Protein Kinases; D011493:Protein Kinase C; D002118:Calcium