Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain.

Abstract

Cytosolic phospholipase A, (cPLAJ associates with natural membranes in response to physiological increases in Ca2+, resulting in the selective hydrolysis of arachidonyl phospholipids.The isolation and sequence analysis of cPLA, cDNA clones from four different species revealed several highly conserved regions.The N H 2 - terminal conserved region is homologous to several other Ca2+-dependent lipid-binding proteins.Here we report that the first 178 residues of c P w , containing the homologous Ca2+-dependent lipid-binding (CaLB) motif, and another recombinant protein containing the cPLAJ1-178) fragment placed at the COOH terminus of the maltose-binding protein (MBP-CaLB) associate with membranes in a Ca2+-dependent manner.cPLA, and MBP-CAB also bind to synthetic liposomes at physiological Ca2+ concentrations, demonstrating that accessory proteins are not required.In contrast, AC2, a truncated cPLA, lacking the CaLB domain, fails to associate with membranes and fails to hydrolyze liposomal substrates.However, both AC2 and cPLA, hydrolyze monomeric 1-palmitoyl-2-lysophosphatidylcholine at identical rates in a Ca2+-independent fashion.These results delineate two functionally distinct domains of cP-, the Ca2+-independent catalytic domain, and the regulatory CaLB domain that presents the catalytic domain to the membrane in response to elevated Ca2+.Phospholipase 4 (PLAJ' releases arachidonic acid from the sn-2 position of membrane glycerophospholipids to initiate the production of prostaglandins and leukotrienes (1, 2).The lyso-* The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact. to the GenBankm/EMBL Data Bank with accession number(s) U10329The nucleotide sequence(s1 reported in this paper has been submitted and U10330.