MECHANISMS OF PHOSPHOLIPASE A2 REGULATION

本研究项目的长期目标是了解 磷脂酶A2（PLA 2）的细胞调节的分子机制。 PLA 2是木兰素生物合成中的第一个酶， 白三烯和血小板活化因子。PLA 2活性过高， 与包括类风湿性关节炎在内的多种疾病有关 炎性充血、与烧伤相关的炎性问题以及 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，脱髓鞘 感染性休克和感染性流产（见参考文献12）。其他方面 炎症，如疼痛和发热以及一些过敏症状 也可以通过调节PLA 2活性来控制。一个 了解PLA 2的细胞调节机制， 大大有助于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PLA 2调节的各种可能方面。这 该模型将包括使用单体（asp-49）PLA 2从毒液 美洲水腹蛇莫卡辛（Agkistrodon piscivorus piscivorus） 和单层磷脂囊泡。特别是膜结构 将测定快速PLA 2活化所需的性质。各种 膜结构的荧光探针将被用来监测时间-- 与PLA 2激活相关的依赖性变化。的三种模式 可以促进明显瞬时PLA 2激活的囊泡将被 从结构的角度仔细研究，试图开发一个 一套快速激活PLA 2的膜结构标准。这些模型 将是小半径的单层囊泡，大单层囊泡 经受渗透压休克和含有 溶血磷脂和脂肪酸的临界浓度（这将 可能作为一个稳定的类似物的结构变化发生在 磷脂酶A2激活过程中的脂质囊泡和囊泡 水解）。最后，可能的PLA 2细胞调节剂，如 甘油二酯或某些多肽（噻虫嗪、心脏毒素、蜂毒肽） 将检测它们诱导PLA 2活化的能力。的 这种激活和可能诱导的膜之间的关系 结构性变化将确定。本项目的方法将 主要涉及酶活性测定（pH stat）和荧光 谱差示扫描量热法，电子显微镜和 [3lP]必要时使用NMR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.