Ulevitch R J, Watanabe Y, Sano M, Lister M D, Deems R A, Dennis E A
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
J Biol Chem. 1988 Mar 5;263(7):3079-85.
The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.
膜磷脂释放游离花生四烯酸被认为是巨噬细胞等炎症细胞中前列腺素、白三烯及相关代谢产物生成过程中的限速步骤。我们之前在巨噬细胞样细胞系P388D1中鉴定出几种不同的磷脂酶，它们可能能够控制花生四烯酸的释放。其中，一种膜结合的、最适pH为碱性、依赖Ca2+的磷脂酶A2特别受关注，因为这种调节酶很可能具有这些特性。现在，这种磷脂酶A2已用辛基葡糖苷从膜组分中溶解并部分纯化。该纯化过程的前两步是丁醇萃取，得到一种冻干的、稳定的磷脂酶A2制剂，且缺乏其他磷脂酶活性。无论底物二棕榈酰磷脂酰胆碱是以超声处理的囊泡形式存在，还是与非离子表面活性剂Triton X-100形成混合胶束，在甘油存在下测定时，这种磷脂酶A2都表现出明显更高的活性。甘油（70%）会增加两种底物形式的Vmax和Km，对于囊泡，Vmax约为15 nmol min-1 mg-1，表观Km约为60 μM；对于混合胶束，Vmax约为100 nmol min-1 mg-1，表观Km约为1 mM。囊泡的Vmax/Km略大于混合胶束。该酶的冻干制剂通常纯化约60倍，适用于评估磷脂酶A2抑制剂，如 manoalide类似物。纯化的后续步骤是乙腈萃取，然后在Aquapore BU-300柱和Superose 12柱上进行高效液相色谱。这使得膜结合的磷脂酶A2得到2500倍的纯化，回收率为25%，对混合胶束中1
