DELINEATION OF 2 FUNCTIONALLY DISTINCT DOMAINS OF CYTOSOLIC PHOSPHOLIPASE-A(2), A REGULATORY CA2-DEPENDENT LIPID-BINDING DOMAIN AND A CA2+-INDEPENDENT CATALYTIC DOMAIN()

Cytosolic phospholipase A(2) (cPLA(2)) associates with natural membran es in response to physiological increases in Ca2+, resulting in the se lective hydrolysis of arachidonyl phospholipids, The isolation and seq uence analysis of cPLA(2) cDNA clones from four different species reve aled several highly conserved regions. The NH2-terminal conserved regi on is homologous to several other Ca2+-dependent lipid-binding protein s. Here we report that the first 178 residues of cPLA(2), containing t he homologous Ca2+-dependent lipid-binding (CaLB) motif, and another r ecombinant protein containing the cPLA(2)(1-178) fragment placed at th e COOH terminus of the maltose-binding protein (MBP-CaLB) associate wi th membranes in a Ca2+-dependent manner. cPLA(2) and MBP-CaLB also bin d to synthetic liposomes at physiological Ca2+ concentrations, demonst rating that accessory proteins are not required. In contrast, Delta C2 , a truncated cPLA(2) lacking the CaLB domain, fails to associate with membranes and fails to hydrolyze liposomal substrates. However, both Delta C2 and cPLA(2) hydrolyze monomeric 1-palmitoyl-2-lysophosphatidy lcholine at identical rates in a Ca2+-independent fashion. These resul ts delineate two functionally distinct domains of cPLA(2), the Ca2+-in dependent catalytic domain, and the regulatory CaLB domain that presen ts the catalytic domain to the membrane in response to elevated Ca2+.