Cloning and expression of a human 14-3-3 protein mediating phospholipolysis. Identification of an arachidonoyl-enzyme intermediate during catalysis.

Abstract

The major phospholipase A2 activity in sheep platelets is mediated by at least three chromatographically resolvable isoforms of a 30-kDa dimeric polypeptide which are responsive to physiologic increments in calcium ion and possess a dramatic substrate selectivity (Loeb, L. A., and Gross, R. W. (1986) J. Biol. Chem. 261, 10467-10470). Herein, we describe the cloning and expression of the human equivalent of one such isoform and demonstrate that it catalyzes the cleavage of the sn-2 fatty acid of choline and ethanolamine glycerophospholipids through the formation of a stable acyl-enzyme intermediate. Transesterification of the sn-2 acyl group of phosphatidylcholine to the recombinant 30-kDa polypeptide is over 50-fold selective for arachidonic acid, is augmented by calcium ion, and results in the formation of an arachidonoyl-thioester intermediate. Homology analysis demonstrated that the polypeptide mediating this transesterification is one member of a family of proteins collectively designated as 14-3-3 proteins. These results demonstrate that at least one intracellular mammalian phospholipase A2 employs a catalytic strategy distinct from that utilized by extracellular phospholipases A2 (i.e. formation of an acyl-enzyme intermediate by nucleophilic attack versus activation of a water molecule) and that arachidonic acid in endogenous phospholipid storage depots can, in principle, be sequentially transferred through an acyl-enzyme intermediate without the prior obligatory release of free arachidonic acid.