BIOCHEMISTRY OF THE ACTIVATION OF PHOSPHOLIPASE A2

磷脂酶 A2 激活的生物化学

• 批准号: 3308872
• 负责人: JOHN D BELL
• 金额: $ 11.31万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308872
• 关键词: acylation; calorimetry; diacylglycerols; electron microscopy; enzyme activity; enzyme induction /repression; fluorescence spectrometry; lipid bilayer membrane; lipolysis; lysolecithins; membrane structure; phospholipase A2; platelets

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.

该研究项目的长期目标是了解 磷脂酶A2（PLA2）调节的分子机制。 pla2是 前列腺素的生物合成中的第一个酶，白细胞，， 来自磷脂前体的血栓烷和血小板激活因子。 多余的PLA2活动与多种疾病有关 炎症性疾病，心血管疾病和几种感染性 疾病。炎症的其他方面，例如疼痛和发炎以及一些 也可以通过调节来控制过敏表现 PLA2活性。 了解活动的机制 在各种PLA2（例如细胞内和细胞外）中，可能 受到调制将极大地帮助设计治疗的策略 这些疾病。 这项工作将侧重于PLA2调节的提议的一种机制： PLA2可以通过特定变化激活的假说 生物膜的结构和动力学。 pla2从 Agkistrodon piscivorus piscivorus和双层的毒液 磷脂酰胆碱和/或磷脂酰甘油将用于大多数 研究。这种酶与 细胞外PLA2与几种炎性疾病状态相关。 由于酶被磷脂水解产物激活，所以 脂肪酸和溶物磷脂，将研究这些药物作为模型 膜的分子如何激活PLA2。这 每个分子与pla2以及双层的相互作用 将检查每种在PLA2激活动力学上的具体作用 在几个实验条件下，将与 这些药物对双层的特性的影响。关系 钙，流动性，电荷和异质性的成分 双层对反应产物诱导的PLA2的激活将 也可以调查。 一旦假设在模型中明确定义 系统，其一般适用性将使用Platelet Pla2和 通过类似的表征，被认为激活PLA2的代理 生理学（二酰基甘油）。该项目中使用的方法将 包括酶活性测定和蛋白质荧光光谱。 代理对膜结构的影响的表征将 包括荧光探针和电子显微镜的光谱。在 总而言之，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。