Isolation and characterization of a phospholipase A, from an inflammatory exudate

Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phosphoethanolamine) and phospholipids of autoclaved Escherichza coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca2+-dependent; Mg2+ and monovalent cations (Na+ and K+) did not substitute for Ca2+ in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[ l-'4C]palmitoyl-2-acylsn-glycero-3-phosphoethanolamine to form only radioactive lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A2 specificity. The phospholipase A2 was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex (275) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDSpolyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (PI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase Az of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed. Supplementary key words phospholipid . Ca2+ dependence . positional specificity * molecular size * cationic protein . polymorphonuclear leukocyte . serum Phospholipases (PLA) accumulate in the peritoneal fluid of patients with cirrhosis and acute pancreatitis (1). PLA reaction products, in particular lysophospholipids, are powerful cytotoxic substances and produce intense inflammation when applied locally (2) or intraperitoneally (3, 4). Furthermore, it has been shown that a variety of commonly used adjuvants increase lysophospholipid production in areas of inflammation ( 5 ) , and that lysophospholipase injected into an inflamed site has potent anti-inflammatory activity, presumably due to its ability to degrade lysophospholipid (5). These data suggest a possible role for PLA in the pathogenesis of inflammatory diseases; but at this time little is known concerning the appearance, origin, or characteristics of PLA in inflammatory fluids. To begin to study the possible role of PLA in the inflammatory process, we have purified and characterized a cell-free PLA2 from rabbit peritoneal fluid. This enzyme closely resembles the granuleassociated PLAz of polymorphonuclear leukocytes found in rabbit peritoneal exudates that we have recently described (6, 7). MATERIALS AND METHODS