Abstract
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 Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) 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 A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed.
Highlights
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 role for PLA in the pathogenesis of inflammatory diseases; but at this time little is known concerning coli
PLA activity of peritoneal supernatant fluid (PSF) and purified CM fractions was consistently linear with respect to time and protein content when phospholipid hydrolysis of 2.5 x lo[8] autoclaved E . coli cells was less than 20% (Fig. 3)
The considerable deviation from linearity that occurred when the hydrolysis was greater than 20% is probably due to limited accessibility of phospholipid within the E. coli envelope, which remains largely intact despite autoclaving
Summary
Polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction. When pure phosphatidylethanolamine was (10,000 cpm added as an ultrasonic suspension in used as substrate, reaction mixtures in a total water), 100 mM Tris-HC1 buffer, pH 7.5, 10 mM volume of 1.0 ml contained 400 nmol of 1-[1-l4C1- CaC12, and protein as indicated. Reaction mixtures palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine were incubated for 30 min at 37°C; the reaction was stopped with two volumes of methanol and lipids were extracted by the method of Bligh and Dyer (9). Phospholipase A activity (l-"C-labeled free fatty acid released from [1-%]oleate-labeled E. coli) was calculated as the percentage of the total E. coli lipid radioactivity and can be converted to nmol of free fatty acid released by assuming that 2.5 X 108E.coli cells contain 5 nmol of phospholipid (6).
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