Journal of Lipid Research, Vol. 19, 18-23, January 1978
Copyright © 1978 by Lipid Research, Inc.

Isolation and characterization of a phospholipase A₂ from an inflammatory exudate

R. Franson , R. Dobrow , J. Weiss , P. Elsbach , and W. B. Weglicki

Department of Biophysics, Medical College of Virginia, Richmond, VA 23298 and Department of Medicine, New York University School of Medicine, New York, NY 10016

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-phospho-ethanolamine) and phospholipids of autoclaved Escherichia 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-[1-¹⁴C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active 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 G75) 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 SDS-polyacrylamide 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 A₂ 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

Submitted on February 15, 1977
Accepted on July 22, 1977

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