CHARACTERIZATION OF HUMAN LEUKOTRIENE C4 SYNTHASE

本申请的拟议目标涉及对 半胱氨基白三烯类化合物的研究 生物合成。这项工作对人的高反应性有重大影响 航空公司，特别是哮喘，超过300万人 美国人。半胱氨基白三烯(白三烯C4、D4和E4)为 脂类介质参与了本病的发病机制。 白三烯C4合成酶催化第一步 白三烯共轭生成半胱氨基白三烯 (LT)A4与还原型谷胱甘肽(GSH)形成白三烯C4。的目标是 这项建议是为了表征LTC4合成酶。全长的cDNA3 在COS-7细胞和sf9昆虫细胞中表达，产生大量 生物活性酵素的量。从这些来源中，这种酶将 纯化及其生化特性的动力学研究 分析并与天然酶进行了比较。站点定向 二十烷类化合物结合部位和活性部位的突变将 阐明特定氨基酸在催化反应中的作用 机制。已经提出了多克隆抗体来研究亚细胞。 细胞分布和LTC4合酶的分布。 正常肺和哮喘肺。这项提案的最终目标将是 克隆人类LTC4合成酶基因，这将使未来 基因组调控的研究。

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.