ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 241, No. 2, September, pp. 413-41’7, 1985 Calcium-Dependent Binding of Calmodulin to Phospholipase A2 Subunits Induces Enzymatic Activation NATHAN MOSKOWITZ,’ ANTONIO ANDRl%,2 WALTER SILVA, LAWRENCE SHAPIRO, WILLIAM SCHOOK, AND SAUL PUSZKIN3 1abratcx-y of Molecular and Cellular Pathology, Department of Pathology, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, New York 10029 Received November 8, 1984, and in revised form May 2, 1985 Calmodulin interacted with phospholipase Az from two different sources, as estab- lished by affinity chromatography, dimethylsuberimidate protein crosslinking, and phospholipase Az assays. Calmodulin was covalently crosslinked to pancreatic and bee venom phospholipases Az in a calcium-dependent manner, and enhanced the enzymatic activities of these phospholipases. Pancreatic phospholipase Az was separated into two species of identical molecular weight by calmodulin affinity chromatography; the species that bound to immobilized calmodulin in a calcium-dependent manner was stimulated by calmodulin. This presents further evidence that phospholipase Az is directly activated by calmodulin. o 1985 Academic PWSS, I~C. Phospholipase A2 (PLAz)4 catalyzes the hydrolysis of the ester bond in the C-2 position of 1,2-sn-phosphoglycerides. This enzyme is present in the mammalian plasma membrane of a variety of cells and tissues and plays a significant role in lipid metabolism (1). PLAzs purified from various sources have been utilized as probes for membrane structure and func- tion (2), and the complete amino acid sequence has been reported for the en- zymes purified from porcine pancreas (3), bee venom (4), and other animal species (5). The various PLAzs exhibit extensive sequence homology, conservation of disul- fide bond placement, and preservation of i Present address: The Johns Hopkins University School of Medicine, Department of Neurosurgery, Baltimore, Md. ‘Fulbright Fellow from the Ministerio de Univ- ersidades e Investigation, Madrid, Spain. a To whom correspondence should be addressed. 4 Abbreviations used: PLAa, phospholipase Aa; CaM, calmodulin; DMS, dimethylsuberimidate; SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(@- aminoethyl ether)-N,N’-tetraacetic acid. many domains of functional importance in the enzymes thus far sequenced (6). An active site containing a histidine residue is considered important for the hydrolytic activity of these enzymes because p-bro- mophenacylbromide inhibits their activi- ties by alkylating this residue (5-8). Calmodulin (CaM), the ubiquitous cal- cium-binding protein in cells (9), has been implicated in the stimulation of platelet and brain synaptic vesicle membranes PLAzs (10, 11) as well as PLAz purified from Naja nuja venom (12). Our approach was to establish whether other purified enzymes had binding affinities for CaM and whether this could be demonstrated unequivocally in an in vitro system. MATERIALS AND METHODS Materials. Dimethylsuberimidate (DMS) was ob- tained from Pierce Chemical. a-Stearoyl-fl-[‘%I-ar- achidonyl-L-phosphatidylcholine (specific activity, 54 mCi/mmol) was purchased from Amersham. Sodium dodecyl sulfate (SDS) and polyacrylamide were from Bio-Rad Laboratories. Silica-G chromatogram sheets (loo-Km thick) were from Fisher Scientific. All other reagents were of analytical grade I. 413 0003-9861185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.