Ceramide 1-Phosphate Acts as a Positive Allosteric Activator of Group IVA Cytosolic Phospholipase A2α and Enhances the Interaction of the Enzyme with Phosphatidylcholine

摘要: Abstract Previous findings from our laboratory have demonstrated that cPLA2α is directly activated by the emerging bioactive sphingolipid, ceramide 1-phosphate (C-1-P) (1). In this study, a Triton X-100/phosphatidylcholine (PC) mixed micelle assay was utilized to determine kinetics and specificity of lipid-enzyme interaction. Using assay, addition C-1-P induced dramatic increase in activity (>15-fold) with Ka 2.4 mol % C-1-P/Triton X-100 micelle. This activation highly specific as other lipids had insignificant effects on activity. Studies using surface-dilution revealed no effect Michaelis-Menten constant, KmB, but decreased dissociation constant (K As) value 87%. Thus, not only increases membrane affinity also may act an allosteric activator enzyme. Surface plasmon resonance analysis C-1-P/cPLA2α interaction verified decrease demonstrating bound PC vesicles containing increased (5-fold) compared alone. The rate found be lipid-specific exception phosphatidylinositol 4,5-bisphosphate, which caused modest vesicle (2-fold). Lastly, binding site for determined within C2-domain cPLA2α, unlike 4,5-bisphosphate. These data demonstrate novel suggest function recruit intracellular membranes well allosterically activate membrane-associated