Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.
Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.
Gehm
B D
BD
Department of Biochemistry, Michigan State University, East Lansing 48824.
Pinke
R M
RM
Laquerre
S
S
Chafouleas
J G
JG
Schultz
D A
DA
Pepperl
D J
DJ
McConnell
D G
DG
D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D002417:Cattle; D004789:Enzyme Activation; D006220:Haloperidol / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D002147:Calmodulin; D007093:Imidazoles; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D013449:Sulfonamides; D014268:Trifluoperazine; D003189:p-Methoxy-N-methylphenethylamine; C031938:calmidazolium; C017967:W 7; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D006220:Haloperidol