MECHANISMS OF PHOSPHOLIPASE A2 REGULATION

这项研究项目的长期目标是了解 磷脂酶A2(PLA2)细胞调控的分子机制 PLA2是前列腺素生物合成的第一种酶， 白三烯和血小板活化因子。过量的PLA2活性是 与包括类风湿性关节炎在内的多种疾病有关 炎症性充血、与烧伤相关的炎性问题和 创伤，湿疹，牛皮癣，胰腺炎，锥虫病，阿米巴病， 心肌梗死、缺血、肺泡蛋白沉积症、脱髓鞘 疾病、感染性休克和感染性流产(见参考文献12)。其他方面 炎症，如疼痛、发热和一些过敏表现 也可以通过调节PLA2的活性来控制。一个 了解PLA2的细胞调节机制将会 极大地帮助设计治疗这些疾病的策略。 本申请中提出的工作将开发一个模型系统 研究细胞内PLA2调节的各个可能方面。这 模型将包括使用来自猪瘟毒液的单体(asp-49)PLA2 美国白腹蛇(Agkistrodon Pisciorus Pisciorus) 和单层磷脂小泡。具体地说，膜结构 将确定快速激活PLA2所需的特性。五花八门 膜结构的荧光探针将用于监测时间-- 与PLA2激活相关的依赖性变化。三种型号的 表面上可以促进PLA2瞬间激活的囊泡将是 从结构的角度仔细研究，试图开发一种 一套快速激活PLA2的膜结构标准。这些型号 会是半径小的单板层囊泡，大的单板层囊泡 出现渗透性休克和大的单层囊泡 溶血磷脂和脂肪酸的临界浓度(将 据推测，它可以稳定地模拟发生在 磷脂酶A2激活过程中的脂泡和囊泡 水解法)。最后，PLA2可能的细胞调节因子，如 甘油二酯或某些多肽(硫氨酸、心脏毒素、蜂毒素) 将检测它们诱导PLA2激活的能力。这个 这种激活与可能的诱导膜的关系 结构性变化将得到确定。该项目的方法将 主要涉及酶活性测定(pH静态)和荧光 光谱学。差示扫描量热法、电子显微镜和电子显微镜 [3LP]如有必要，将使用核磁共振来表征囊泡结构。

The long-term objective of this research project is to understand the molecular mechanisms of cellular regulation of phospholipase A2 (PLA2). PLA2 is the first enzyme in the biosynthesis of prostaglandins, leukotrienes and platelet activating factor. Excess PLA2 activity is implicated in a number of diseases including rheumatoid arthritis inflammatory hyperemia, inflammatory problems associated with burns and trauma, eczema, psoriasis, pancreatitis, trypanosomiasis, amoebiasis, myocardial infarction, ischemia, alveolar proteinasis, demyelinating disease, septic shock and infectious abortions (see Ref. 12). Other aspects of inflammation such as pain and pyrexia and some allergic manifestations could also conceivably be controlled by regulating PLA2 activity. An understanding of the mechanisms of cellular regulation of PLA2 would greatly aid in the design of strategies for treating these disorders. The work proposed in this application will develop a model system for investigating various possible aspects of cellular PLA2 regulation. This model will include the use of the monomeric (asp-49) PLA2 from the venom of the American Cottonmouth Water Moccasin (Agkistrodon piscivorus piscivorus) and unilamellar phospholipid vesicles. Specifically the membrane structural properities required for rapid PLA2 activation will be determined. Various fluorescent probes of membrane structure will be used to monitor time-- dependent changes that correlate with PLA2 activation. Three models of vesicles that can promote apparently instantaneous PLA2 activation will be carefully examined from a structural perspective in attempt to develop a set of membrane structural criteria for rapid PLA2 activation. These models will be unilamellar vesicles of small radius, large unilamellar vesicles submitted to osmotic shock and large unilamellar vesicles containing a critical concentration of lysophospholipid and fatty acid (which will presumably serve as a stable analog of the structural changes occurring in lipid vesicles during the time course of PLA2 activation and vesicle hydrolysis). Finally, possible cellular regulators of PLA2 such as diacylglycerol or certain polypeptides (thionine, cardiotoxin, mellitin) will be examined for their ability to induce PLA2 activation. The relationship between such activation and possible induced membrane structural changes will be determined. Methodology for this project will primarily involve enzyme activity assay (pH stat) and fluorescence spectroscopy. Differential scanning calorimetry, electron microscopy and [3lP] NMR will he used as necessary to characterize vesicle structure.