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Journal of Lipid Research, Vol. 48, 2701-2708, December 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Anionic lipids activate group IVA cytosolic phospholipase A₂ via distinct and separate mechanisms

Preeti Subramanian^*, Mohsin Vora, Luciana B. Gentile^*, Robert V. Stahelin^1,2,, and Charles E. Chalfant^1,2,*,**,

^* Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0614
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN 46617
Department of Chemistry and Biochemistry and Walther Center for Cancer Research, University of Notre Dame, South Bend, IN 46617
^** Research and Development, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, VA 23249
The Massey Cancer Center, Richmond, VA 23298

Published, JLR Papers in Press, September 21, 2007.

¹ R. V. Stahelin and C. E. Chalfant contributed equally to this article.

² To whom correspondence should be addressed. e-mail: rstaheli{at}iupui.edu (R.V.S.); cechalfant{at}vcu.edu (C.E.C.)

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A₂ (cPLA₂). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P₂ in decreasing the dissociation constant of the cPLA₂-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P₂, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P₂ activated cPLA₂ with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P₂, but not C1P, increased the penetration ability of cPLA₂ into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA₂ by anionic lipids. First, C1P activates cPLA₂ by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P₂ activates the enzyme by increasing catalytic efficiency through increased membrane penetration.

Supplementary key words ceramide-1-phosphate • ceramide kinase • eicosanoids • prostaglandins • inflammation • surface dilution kinetics • surface plasmon resonance • phosphatidylinositol-4,5-bisphosphate

Abbreviations: AA, arachidonic acid; cPLA₂, group IVA cytosolic phospholipase A₂; C1P, ceramide-1-phosphate; K_d, dissociation constant; OPPC, 1-oleoyl-2-palmitoyl-phosphatidylcholine; PAPC, 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine; PC, phosphatidylcholine; PI(4,5)P₂, phosphatidylinositol-4,5-bisphosphate; SPR, surface plasmon resonance

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