Regulatory aspects of rat liver mitochondrial phospholipase A2: effects of calcium ions and calmodulin.
A comparative study was made of the metal ion requirement of rat liver mitochondrial phospholipase A2 in purified and membrane-associated forms. Membrane-bound enzyme was assayed using either exogenous or endogenous phosphatidylethanolamine. Although several divalent metal ions caused increased activity of the membrane-associated enzyme, only Ca2+ and Sr2+ activated the purified phospholipase A2. The activity in the presence of Sr2+ amounted to about 25% of that found with Ca2+. When the Ca2+ concentration was varied two activity plateaus were observed. The corresponding dissociation constants varied from 6 to 20 microM Ca2+ and from 1.4 to 12 mM Ca2+ for the high- and low-affinity binding sites, respectively, depending on the assay conditions and whether purified or membrane-bound enzyme was used. A kSr2+ of 60 microM was found for the high-affinity binding site. The effect of calmodulin and its antagonist trifluoperazine was also investigated using purified and membrane-associated enzyme. When membrane-bound enzyme was measured with exogenous phosphatidylethanolamine, small stimulations by calmodulin were found. However, these were not believed to indicate a specific role for calmodulin in the Ca2+ dependency of the phospholipase A2, since trifluoperazine did not lower the activity of the membrane-bound enzyme to levels below those found in the presence of Ca2+ alone. Membrane-bound enzyme in its action toward endogenous phosphatidylethanolamine was neither stimulated by calmodulin nor inhibited by trifluoperazine. Purified enzyme was also not stimulated by calmodulin, while trifluoperazine caused small stimulations, presumably due to interactions at the substrate level. These results indicate that calmodulin involvement in phospholipase A2 activation should not be generalized.
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002413:Cations, Divalent / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation / Q000187:drug effects; D008930:Mitochondria, Liver / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D002413:Cations, Divalent; D014268:Trifluoperazine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium