NADPH-DEPENDENT METABOLISM OF ARACHIDONIC ACID

花生四烯酸的 NADPH 依赖性代谢

• 批准号: 2179039
• 负责人: JORGE CAPDEVILA
• 金额: $ 24.51万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179039
• 关键词: NAD(H) phosphate; arachidonate; biological signal transduction; calcium flux; cytochrome P450; eicosanoid metabolism; eicosanoids; enzyme activity; enzyme reconstitution; laboratory rabbit; laboratory rat; liver cells; microsomes; molecular cloning; nucleic acid hybridization; nucleic acid sequence; oxygenases; protein isoforms; recombinant proteins; stereoisomer; tissue /cell culture

The oxygenated metabolism of arachidonic acid is characterized by the increasing complexity of its metabolic pathways and products and, by the diversity of functional properties attributed to them. While the metabolic versatility of the arachidonate molecular template allows for the storage and transfer of an exceptional degree of functionally meaningful chemical information, the integration of these multiple reactions and of their associated products and biological activities to cell, tissue or body physiology is an important task which, although difficult and complex, is greatly facilitated by the understanding of the biochemistry of the reactions, the structure of metabolites and the molecular properties of the enzymes involved. The microsomal cytochrome P-450 arachidonic acid epoxygenase catalyzes the enantioselective formation of four regioisomeric epoxyeicosatrienoic acids (EETs). The asymmetric nature of the EETs present, endogenously, in several tissues demonstrated their enzymatic origin and confirmed cytochrome P450 as the in vivo arachidonic acid epoxygenase and, as a formal member of the fatty acid metabolic cascade. The last few years have seen a renewed interest in the delineation of the role of cytochrome P-450 in the bioactivation of arachidonic acid and, therefore, in its significance for cell and organ physiology. Many of these efforts have been stimulated by the potent biological activities of the EETs as mediators for peptide hormone release, vasoactive molecules and modulators of membrane ion fluxes. More recently, several lines of evidence suggest that this pathway may be involved, in yet undefined ways, in the pathophysiology of hypertension. The potential physiological and clinical implication of this proposal are of pivotal importance and area therefore, stimulating extensive research. During the last 10 years, this project completed a detailed characterization of the enzymology and the structural properties of the epoxygenase metabolites, those studies contributed substantially to our present understanding of the biochemical and functional significance of this pathway. Further progress will require a detailed molecular knowledge of the enzymes involved, as well as the development of the biospecific probes needed for studies of the regulation of this pathway at the protein and gene level. We propose to utilize modern molecular biology techniques in order to identify and characterize the cytochrome P-450 isoforms involved in the generation of endogenous EETs. We propose to clone the relevant gene(s), to express the cloned cDNA to characterize the recombinant protein(s), to generate immunospecific probes and to study the role of recombinant proteins in cell EET and calcium metabolism.

蛛网膜酸的氧化代谢的特征是 提高其代谢途径和产品的复杂性，以及 功能属性的多样性归因于它们。而代谢 蛛网分子模板的多功能性允许存储 并转移特殊程度有意义的化学物质 信息，这些多种反应的整合及其 相关的产品和生物活性与细胞，组织或身体 生理学是一项重要任务，尽管困难且复杂，但 理解的生物化学极大地促进了 反应，代谢物的结构和分子特性 涉及的酶。微粒体细胞色素P-450蛛网膜酸 环氧酶催化四个区域异构体的对映选择性形成 环氧树钠酸（EET）。 EET的不对称性质 在几个组织中，内源性地存在酶促的 起源并确认为体内花生四烯酸为细胞色素P450 环氧酶，作为脂肪酸代谢级联反应的正式成员。 在过去的几年中，人们对划定的兴趣重新兴趣 细胞色素p-450在花生四烯酸生物活化中的作用，以及 因此，它对细胞和器官生理学意义。许多 这些努力受到了强大的生物学活动的刺激 EET作为肽激素释放的介体，血管活性分子 和膜离子通量的调节剂。最近，几行 有证据表明，该途径可能以未定义的方式参与 在高血压的病理生理学中。潜在的生理和 该提案的临床意义至关重要，面积很重要 因此，刺激广泛的研究。 在过去的十年中，该项目完成了详细的 酶学的表征和结构特性 环氧酶代谢产物，这些研究为我们的 当前对生化和功能意义的理解 这条路。进一步的进步将需要详细的分子知识 涉及的酶以及生物特异性的开发 研究该途径在蛋白质上所需的探针 和基因水平。我们建议利用现代分子生物学技术 为了识别和表征细胞色素P-450同工型 参与内源性Eets的产生。我们建议克隆 相关基因表达克隆的cDNA以表征 重组蛋白，以产生免疫特异性探针并研究 重组蛋白在细胞EET和钙代谢中的作用。