Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin

Biochemistry. 1991 Nov 26;30(47):11302-6. doi: 10.1021/bi00111a016.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Calmodulin / antagonists & inhibitors*
  • Calmodulin / pharmacology
  • Cattle
  • Enzyme Activation
  • Haloperidol / pharmacology
  • Imidazoles / pharmacology
  • Kinetics
  • Phosphatidylinositol 4,5-Diphosphate
  • Phosphatidylinositol Phosphates
  • Phosphatidylinositols / metabolism
  • Phosphoinositide Phospholipase C
  • Phosphoric Diester Hydrolases / metabolism*
  • Rod Cell Outer Segment / enzymology*
  • Substrate Specificity
  • Sulfonamides / pharmacology
  • Trifluoperazine / pharmacology
  • Type C Phospholipases / metabolism*
  • p-Methoxy-N-methylphenethylamine / pharmacology

Substances

  • Calmodulin
  • Imidazoles
  • Phosphatidylinositol 4,5-Diphosphate
  • Phosphatidylinositol Phosphates
  • Phosphatidylinositols
  • Sulfonamides
  • Trifluoperazine
  • p-Methoxy-N-methylphenethylamine
  • calmidazolium
  • W 7
  • Phosphoric Diester Hydrolases
  • Type C Phospholipases
  • Phosphoinositide Phospholipase C
  • Haloperidol