REGULATION OF 85KDA PHOSPHOLIPASE A2 ACTIVATION

本应用的目的是研究其生化特性。 和花生四烯酰基水解型磷脂酶的调节机制 小鼠巨噬细胞系RAW 264.7的A2(PLA2)。制作 在生物活性脂质介体中，二十烷类化合物与血小板活化 因子，是由炎症细胞中的PLA2激活而启动的。 尽管PLA2在炎性脂质的产生中起着重要作用 介体，人们对这种酶的性质知之甚少。A钙 需要，从巨噬细胞中纯化出高分子量的PLA2 细胞系，RAW 264.7。这种酶表现出独特的性质，包括 SN-2花生四烯酸的特异性，提示其在脂质介质中的作用 制作。PLA2还具有类似于两亲性的性质， 钙/磷脂结合蛋白，如蛋白激酶C。 阴离子磷脂刺激磷脂酶A2的活性 二酰甘油和阴离子磷脂降低钙 酶的完全活性所需的浓度。此外， 磷脂酶A2存在于胞浆中，但表现出钙依赖关系。 有膜的。这些特性可能在PLA2的调节中发挥作用 活体内活动。 这项提案的一个具体目标是刺激264.7的原始人口 并确定巨噬细胞的活性是否增加 花生四烯酸水解磷脂酶A2，无论它是否转移到膜和 这些事件是否与细胞内钙离子的变化有关 集中精神。特异性膜脂在膜调节中的作用 磷脂酶A2的结合和酶的底物专一性调节 将使用模型膜在体外进行研究。PLA2是 假定由翻译后修饰来调节，例如 磷酸化、酰化或蛋白水解化过程，这些过程在 调节磷脂酶A2的活性并与膜结合。这些进程将 在体外和体内进行研究。优先考虑的是获得PLA2抗体 对于酶的翻译后修饰的研究和 转位到特定的膜上。最终目标是确定 克隆了PLA2基因的全长序列，推导出其氨基酸序列。 这种酶。这将有助于阐明重要的结构域 与低分子量PLA2酶和TO的同源性比较 两性、钙/磷脂结合蛋白。澄清 脂类相关酶的性质及其调节机制 调解人的产生对于更好地理解潜力至关重要 控制炎症过程的方法。

The objective of this application is to study the biochemical properties and mechanisms of regulation of an arachidonoyl-hydrolyzing phospholipase A2 (PLA2) from the mouse macrophage cell line, RAW 264.7. The production of the bioactive lipid mediators, the eicosanoids and platelet-activating factor, is initiated by the activation of a PLA2 in inflammatory cells. Despite the important role of PLA2 in the production of inflammatory lipid mediators, little is known about the properties of this enzyme. A calcium requiring, high molecular weight PLA2 has been purified from the macrophage cell line, RAW 264.7. This enzyme exhibits unique properties, including specificity for sn-2 arachidonic acid, suggesting a role in lipid mediator production. The PLA2 also has properties similar to the amphitropic, calcium/phospholipid binding proteins, such as protein kinase C. The activity of the PLA2 is stimulated by anionic phospholipids and diacylglycerol; and anionic phospholipids decrease the calcium concentration required for full activity of the enzyme. In addition, the PLA2 is found in the cytosol, but exhibits calcium-dependent association with membrane. These properties may play a role in the regulation of PLA2 activity in vivo. One specific aim of this proposal, is to stimulate the RAW 264.7 macrophages and to determine if there is an increase in activity of the arachidonoyl-hydrolyzing PLA2, whether it translocates to membrane and whether these events correlate with changes in the intracellular calcium concentration. The role of specific membrane lipids in mediating membrane binding of the PLA2 and modulating the substrate specificity of the enzyme will be investigated in vitro using model membranes. The PLA2 is hypothesized to be regulated by postranslational modification, such as phosphorylation, acylation or proteolytic processing, that play a role in modulating PLA2 activity and binding to the membrane. These processes will be studied in vitro and in vivo. A priority is to obtain a PLA2 antibody for studies of posttranslational modification of the enzyme and translocation to specific membranes. The final aim is to determine the sequence of the PLA2 cDNA, in order to deduce the amino acid sequence of the enzyme. This will allow elucidation of important structural domains and homology comparisons to the low molecular weight PLA2 enzymes and to the amphitropic, calcium/phospholipid binding proteins. Elucidating the properties and mechanisms of regulation of enzymes involved in lipid mediator production is critical for a better understanding of potential methods for controlling the inflammatory process.