Leslie C C
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
J Biol Chem. 1991 Jun 15;266(17):11366-71.
The first step in the production of eicosanoids and platelet-activating factor is the hydrolysis of arachidonic acid from membrane phospholipid by phospholipase A2. We previously purified from the macrophage cell line RAW 264.7 an intracellular phospholipase A2 that preferentially hydrolyzes sn-2-arachidonic acid. The enzyme exhibits a molecular mass of 100 kDa and an isoelectric point of 5.6. When assayed for other activities, the phospholipase A2 was found to exhibit lysophospholipase activity against palmitoyllysoglycerophosphocholine, and both activities copurified to a single band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. An antibody against the macrophage enzyme was found to quantitatively immunoprecipitate both phospholipase A2 and lysophospholipase activities from a crude cytosolic fraction. When the immunoprecipitated material was analyzed on immunoblots, a single band at 100 kDa was evident, further suggesting that a single protein possessed both enzyme activities. When assayed as a function of palmitoyllysoglycerophosphocholine concentration and plotted as a double-reciprocal plot, two different slopes were apparent, corresponding to concentrations above and below the critical micellar concentration (7 microM) of the substrate. Above the critical micellar concentration, lysophospholipase exhibited an apparent Km of 25 microM and a Vmax of 1.5 mumol/min/mg. Calcium was not required for lysophospholipase activity, in contrast to phospholipase A2 activity. The enzyme, when assayed as either a phospholipase A2 or lysophospholipase, exhibited nonlinear kinetics beyond 1-2 min despite low substrate conversion. Readdition to more substrate after the activity plateaued did not result in further enzyme activity, ruling out substrate depletion. Readdition of enzyme, however, resulted in another burst of enzyme activity. The results are not consistent with product inhibition, but suggest that the enzyme may be subject to inactivation during catalysis.
类二十烷酸和血小板激活因子产生的第一步是磷脂酶A2从膜磷脂中水解花生四烯酸。我们之前从巨噬细胞系RAW 264.7中纯化出一种细胞内磷脂酶A2，它优先水解sn-2-花生四烯酸。该酶的分子量为100 kDa，等电点为5.6。在检测其他活性时，发现该磷脂酶A2对棕榈酰溶血甘油磷酸胆碱具有溶血磷脂酶活性，并且这两种活性在银染的十二烷基硫酸钠-聚丙烯酰胺凝胶上共纯化到一条带。发现一种针对巨噬细胞酶的抗体能从粗胞质组分中定量免疫沉淀磷脂酶A2和溶血磷脂酶活性。当对免疫沉淀的物质进行免疫印迹分析时，100 kDa处出现一条单一的带，进一步表明单一蛋白质具有这两种酶活性。当以棕榈酰溶血甘油磷酸胆碱浓度为函数进行检测并绘制双倒数图时，出现了两个不同的斜率，分别对应底物临界胶束浓度（7 microM）以上和以下的浓度。在临界胶束浓度以上，溶血磷脂酶的表观Km为25 microM，Vmax为1.5微摩尔/分钟/毫克。与磷脂酶A2活性相反，溶血磷脂酶活性不需要钙。该酶在作为磷脂酶A2或溶血磷脂酶进行检测时，尽管底物转化率较低，但在1 - 2分钟后表现出非线性动力学。活性达到平稳后再添加更多底物并没有导致酶活性进一步增加，排除了底物耗尽的可能性。然而，再添加酶会导致酶活性再次爆发。这些结果与产物抑制不一致，但表明该酶在催化过程中可能会失活。
