CHARACTERIZATION OF HUMAN LEUKOTRIENE C4 SYNTHASE

本申请提出的目的涉及对本发明的方法的研究。 调节半胱氨酰白三烯的研究， 生物合成这项工作对高反应性有重大影响， 气道，特别是支气管哮喘， 美国人半胱氨酰白三烯（白三烯C4、D4和E4）是 脂质介质参与这种疾病的发病机制。 白三烯C4合酶催化的第一个关键步骤， 通过白三烯的结合形成半胱氨酰白三烯 （LT）A4与还原型谷胱甘肽（GSH）形成白三烯C4。的目标 该建议是表征LTC 4合酶。全长cDNA具有 在COS-7细胞和sf 9昆虫细胞中表达， 生物活性酶的量。从这些来源，酶将 用动力学方法研究了其生化特性 分析并与天然酶进行比较。定点 假定的类二十烷酸结合位点和活性位点的诱变将 阐明参与催化的特定氨基酸的作用 机制多克隆抗体已被提出来研究亚细胞 和细胞分布以及LTC 4合酶在 正常和哮喘的肺。本提案的最终目标是 克隆人类LTC 4合酶基因，这将使未来 基因调控的研究。

The proposed objective of this application relates to the study of the regulation of the cysteinyl leukotrienes by investigation their biosynthesis. This work has a significant impact on hyperresponsiveness of airways, particularly bronchial asthma, which acts over 3 million Americans. The cysteinyl leukotrienes (leukotrienes C4, D4, and E4) are lipid mediators implicated in the pathogenesis of this disease. Leukotriene C4 synthase catalyzes the first committed step in the formation of the cysteinyl leukotrienes by the conjugation of leukotriene (LT)A4 with reduced glutathione (GSH) to form leukotriene C4. The goal of this proposal is to characterize LTC4 synthase. The full length cDNA has been expressed in COS-7 cells and sf9 insect cells, yielding substantial amounts of biologically active enzyme. From these sources, the enzyme will be purified and its biochemical characteristics studied by kinetic analysis and compared to that of the native enzyme. Site-directed mutagenesis of the putative eicosanoid binding site and active site will elucidate the role of specific amino acids involved in the catalytic mechanism. Polyclonal antibodies have been raised to study the subcellular and cellular distribution as well as the distribution of LTC4 synthase in normal and asthmatic lungs. The final objective of this proposal will be to clone the human gene for LTC4 synthase, which will permit future studies of genomic regulation.