REGULATION OF 85KDA PHOSPHOLIPASE A2 ACTIVATION

85KDA 磷脂酶 A2 激活的调节

• 批准号: 6202244
• 负责人: CHRISTINA Carroll LESLIE
• 金额: $ 24.23万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202244
• 关键词: arachidonate; enzyme mechanism; fatty acid metabolism; immunofluorescence technique; inflammation; macrophage; mass spectrometry; membrane lipids; phospholipase A2; phosphorylation; protein kinase; protein sequence; protein transport; tissue /cell culture

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.

本申请的目的是研究生物化学性质 花生四烯酸水解磷脂酶的调节机制 A2（PLA 2），来自小鼠巨噬细胞系RAW 264.7。 生产 生物活性脂质介质，类花生酸和血小板活化 因子，是由炎症细胞中PLA 2的活化引发的。 尽管PLA 2在炎症脂质的产生中起重要作用， 介质，对这种酶的性质知之甚少。 钙 从巨噬细胞中纯化出所需的高分子量PLA 2 细胞系RAW 264.7。 这种酶具有独特的特性，包括 对sn-2花生四烯酸的特异性，表明在脂质介质中的作用 生产 PLA 2也具有类似于两亲性的性质， 钙/磷脂结合蛋白，如蛋白激酶C。 的 PLA 2的活性被阴离子磷脂刺激， 甘油二酯;和阴离子磷脂降低钙 该浓度是酶完全活性所需的浓度。 此外该 PLA 2存在于胞质溶胶中，但表现出钙依赖性缔合 膜。 这些特性可能在PLA 2的调节中起作用。 体内活性。 该提案的一个具体目标是刺激RAW 264.7 巨噬细胞，并确定是否有增加的活动， 花生四烯酸水解磷脂酶A2，无论它是否易位到膜， 这些事件是否与细胞内钙的变化相关 浓度. 特异性膜脂在介导膜 PLA 2的结合和调节酶的底物特异性 将使用模型膜进行体外研究。 PLA 2是 假设受翻译后修饰的调节，例如 磷酸化、酰化或蛋白水解加工，在 调节PLA 2活性并与膜结合。 这些进程将 在体外和体内进行研究。 首先是获得PLA 2抗体 用于酶的翻译后修饰的研究， 转移到特定的膜上。 最终目的是确定 PLA 2 cDNA的氨基酸序列，以推导PLA 2 cDNA的氨基酸序列。 酶 这将允许阐明重要的结构域 以及与低分子量PLA 2酶和与 两亲性钙/磷脂结合蛋白。 阐明 脂类相关酶的性质和调节机制 介体的产生对于更好地理解潜在的 控制炎症过程的方法。