Isolation and characterization of a phospholipase A2 from an inflammatory exudate.
Isolation and characterization of a phospholipase A2 from an inflammatory exudate.
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从炎症渗出液中分离和表征磷脂酶 A2。
DOI：
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发表时间：
1978
影响因子：
6.5
通讯作者：
W. Weglicki
中科院分区：
文献类型：
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作者：
R. Franson;R. Dobrow;J. Weiss;P. Elsbach;W. Weglicki
在注射1%糖原的兔体内产生的无菌腹膜渗出物在无细胞上清液中含有磷脂酶A活性，该上清液可降解合成的磷脂(1，2-diacyl-sn-glycero-3-phospho-ethanolamine)和高压灭菌的大肠杆菌的磷脂。该磷脂酶活性(磷脂酰乳糖水解酶EC 3.1.1.4)表现出明显的双峰最适pH(pH 6.0和pH 7.5)，并依赖于Ca(2+)，Mg(2+)和单价阳离子(Na(+)和K(+))不能替代反应中的Ca(2+)，EDTA是一种有效的抑制剂。该磷脂酶对1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine进行水解，生成具有放射性的溶血磷脂酰乙醇胺，表明该酶具有磷脂酶A(2)的专一性。该磷脂酶A(2)经两次连续的羧甲基葡聚糖凝胶层析，纯化了302倍。纯化的酶经Sephadex G75凝胶过滤后，得到一个生物学活性单峰，其相对分子质量约为14,800。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测得的相对分子质量也相同，只有一条带。在pH为4.3的条件下，该组分的聚丙烯酰胺凝胶电泳法显示出一条带染料的单一蛋白质带移出溶菌酶，表明该蛋白质比溶菌酶更碱性(等电点10.5)。这种可溶酶的酶性质和理化性质与最近报道的一种磷脂酶A(2)非常相似，该酶来源于糖原诱导的腹膜渗出物。讨论了该可溶性酶的可能来源和生理作用。
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.