MEMBRANE PENETRATION OF CYTOSOLIC PHOSPHOLIPASE A(2) IS NECESSARY FORITS INTERFACIAL CATALYSIS AND ARACHIDONATE SPECIFICITY

To determine the mechanism of calcium-dependent membrane binding of cy tosolic phospholipase A(2) (cPLA(2)), we measured the interactions of cPLA(2) with phospholipid monolayers and polymerizable mixed liposomes containing various phospholipids. In the presence of calcium, cPLA(2) showed much higher penetrating power than secretory human pancreatic PLA(2) toward anionic and electrically neutral phospholipid monolayers . cPLA(2) also showed ca. 30-fold higher binding affinity for nonpolym erized lipoyloxy)dodecanoyl]-sn-glycero-1-phosphoglycerol (D-BLPG) lip osomes than for polymerized ones where the membrane penetration of pro tein is significantly restricted. Consistent with this difference in m embrane binding affinity, cPLA(2) showed 20-fold higher activity towar d fluorogenic substrates, decyl)-2-arachidonoyl-sn-glycero-3-phosphoch oline, inserted in nonpolymerized D-BLPG liposomes than the same subst rate in polymerized D-BLPG liposomes. Furthermore, cPLA(2) showed much higher sn-2 acyl group specificity (arachidonate specificity) and hea dgroup specificity in nonpolymerized D-BLPG liposomes than in polymeri zed D-BLPG liposomes. Finally, diacylglycerols, such as 1,2-dioleoyl-s n-glycerol, selectively enhanced the membrane penetration, hydrophobic membrane binding, and interfacial enzyme activity of cPLA(2). Taken t ogether, these results indicate the following: (1) calcium not only br ings cPLA(2) to the membrane surface but also induces its membrane pen etration. (2) This unique calcium-dependent membrane penetration of cP LA(2) is necessary for its interfacial binding and substrate specifici ty. (3) Diacylglycerols might work as a cellular activator of cPLA(2) by enhancing its membrane penetration and hydrophobic membrane binding .