CALCIUM-DEPENDENT AND CALCIUM-INDEPENDENT INTERFACIAL BINDING AND CATALYSIS OF CYTOSOLIC GROUP-IV PHOSPHOLIPASE-A(2)

Cytosolic group IV phospholipase A(2) (cPLA2) plays a role in liberati ng arachidonic acid from the sn-2 position of mammalian cellular phosp holipids. The enzyme consists of a catalytic domain joined to an N-ter minal calcium-dependent, membrane binding domain (C2 domain). The inte rfacial binding properties of the full-length, nonphosphorylated enzym e and its C2 domain to phospholipid vesicles were studied as a functio n of vesicle phospholipid composition and calcium concentration. The b inding of cPLA2 to phosphatidylcholine vesicles is mostly governed by its C2 domain; binding is relatively weak, and calcium enhances bindin g and interfacial catalysis by about 10-fold. Catalytically productive interfacial binding was measured by monitoring the increase in the ra te of cPLA2-catalyzed hydrolysis of a fluorimetric substrate present i n vesicles as a function of bulk vesicle concentration. Enzyme-vesicle binding was also measured by fluorescence as was enzyme-calcium bindi ng. Compared to zwitterionic vesicles, cPLA2 binding to anionic phosph atidylmethanol vesicles is of higher affinity and calcium-independent, although calcium is required for the binding of the C2 domain to thes e anionic vesicles. cPLA2 is fully catalytically active on phosphatidy lmethanol vesicles in the absence of calcium. Phosphatidylserine is no t a good replacement for phosphatidylmethanol for inducing high-affini ty, calcium-independent binding of cPLA2, These results reveal two mod es of catalytically productive interfacial binding of cPLA2: calcium-d ependent anchoring via the C2 domain and a calcium-independent compone nt involving a phosphatidylmethanol recognition element in the catalyt ic domain. They also show that membrane binding of cPLA2 is not, in ge neral, predicted by the interfacial binding properties of its C2 domai n.