BIOCHEMISTRY OF THE ACTIVATION OF PHOSPHOLIPASE A2

这项研究项目的长期目标是了解 磷脂酶A2(PLA2)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， 来自磷脂前体的血栓烷和血小板激活因子。 PLA2活性过高与许多疾病有关，包括 炎症性疾病、心血管疾病和几种传染性疾病 疾病。炎症的其他方面，如疼痛和发热以及一些 可以想象，过敏表现也可以通过调节 磷脂酶A2活性。对活动的机制的理解 不同种类的PLA2(例如，细胞内和细胞外)可以 将在很大程度上有助于设计治疗策略 这些障碍。 这项工作将集中在拟议的PLA2调节机制之一： 认为PLA2可能通过细胞内特定的改变而被激活的假说 生物膜的结构和动力学。从鼠脑组织中分离纯化磷脂酶A2 江西产蛇毒液及双层蛇毒 磷脂酰胆碱和/或磷脂酰甘油将用于 这些研究。这种酶在结构上与 细胞外PLA2与多种炎症性疾病状态相关。 由于该酶是由磷脂水解产物激活的， 脂肪酸和溶血磷脂，这些药物将作为模型研究 扰乱细胞膜的分子如何激活PLA2。这个 每个分子与磷脂酶A2以及与双层和 我们将考察它们在PLA2激活动力学中的具体作用 在几个实验条件下，并将与 这些试剂对双分子膜性能的影响。两国关系 钙、流动性、电荷和组成的不均一性 双分子膜对反应产物诱导的PLA2的激活作用 也被调查。一旦假设在模型中被很好地定义 系统，其一般适用性将测试使用血小板PLA2和 通过对被认为激活PLA2的试剂进行类似的表征 生理上(二酰基甘油)。此项目中使用的方法将 包括酶活性测定和蛋白质荧光光谱分析。 表征试剂对膜结构的影响将 包括荧光探针的光谱学和电子显微镜。在……里面 总结一下，这些实验将提供有关 双层结构如何与PLA2活性相关的分子细节。

The long-term objective of this research project is to understand the molecular mechanisms of regulation of phospholipase A2 (PLA2). PLA2 is the first enzyme in the biosynthesis of prostaglandins, leukotrienes, thromboxanes and platelet-activating factor from phospholipid precursors. Excess PLA2 activity is implicated in a number of diseases including inflammatory diseases, cardiovascular disease and several infectious diseases. 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 by which the activity of the various species of PLA2 (e.g. intracellular and extracellular) may be modulated would greatly aid in the design of strategies for treating these disorders. This work will focus on one of the proposed mechanisms of PLA2 regulation: the hypothesis that PLA2 may be activated by specific alterations in the structure and dynamics of biological membranes. PLA2 purified from the venom of Agkistrodon piscivorus piscivorus and bilayers of phosphatidylcholines and/or phosphatidylglycerols will be used for most of the studies. This enzyme exhibits high structural similarity to the extracellular PLA2 associated with several inflammatory disease states. Since the enzyme is activated by the products of phospholipid hydrolysis, fatty acid and lysophospholipid, these agents will be studied as models of how molecules that perturb the membrane may activate PLA2. The interactions of each molecule with PLA2 and with the bilayer and the specific role of each on the kinetics of PLA2 activation will be examined under several experimental conditions and will be correlated with the effects of these agents on the properties of the bilayer. The relationship of calcium, fluidity, charge and heterogeneity of composition in the bilayer to the activation of PLA2 induced by the reaction product will also be investigated. Once the hypothesis is well-defined in the model system, its general applicability will be tested using platelet PLA2 and by similar characterization of an agent thought to activate PLA2 physiologically (diacylglycerol). The methods used in this project will include enzyme activity assay and protein fluorescence spectroscopy. Characterization of the effects of agents on membrane structure will include spectroscopy of fluorescent probes and electron microscopy. In summary, these experiments will provide information concerning the molecular details of how bilayer structure relates to PLA2 activity.