CHARACTERIZATION OF HUMAN LEUKOTRIENE C4 SYNTHASE

人白三烯 C4 合成酶的表征

• 批准号: 6043649
• 负责人: JOHN F PENROSE
• 金额: $ 12.31万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6043649
• 关键词: active sites; antisense nucleic acid; chemical kinetics; enzyme activity; enzyme biosynthesis; enzyme structure; fatty acid synthase; high performance liquid chromatography; human genetic material tag; human tissue; immunocytochemistry; laboratory rabbit; leukotrienes; messenger RNA; polymerase chain reaction; protein purification; site directed mutagenesis; tissue /cell culture; western blottings

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.

本申请的拟议目标涉及研究 通过研究半胱氨酰白三烯的调节 生物合成。这项工作对高反应性有重大影响 呼吸道疾病，尤其是支气管哮喘，影响了超过 300 万人 美国人。半胱氨酰白三烯（白三烯 C4、D4 和 E4）是 脂质介质参与该疾病的发病机制。 白三烯 C4 合酶催化第一个关键步骤 通过白三烯的缀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。目标是 该提案旨在表征 LTC4 合酶。全长 cDNA 有 在 COS-7 细胞和 sf9 昆虫细胞中表达，产生大量 一定量的生物活性酶。从这些来源，酶将 纯化并通过动力学研究其生化特性 分析并与天然酶进行比较。现场定向 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。已产生多克隆抗体来研究亚细胞 和细胞分布以及LTC4合酶的分布 正常肺和哮喘肺。该提案的最终目标是 克隆 LTC4 合酶的人类基因，这将允许未来 基因组调控的研究。

项目成果

5' flanking region polymorphism of the gene encoding leukotriene C4 synthase does not correlate with the aspirin-intolerant asthma phenotype in the United States.

编码白三烯 C4 合酶的基因的 5 侧翼区域多态性与美国阿司匹林不耐受的哮喘表型无关。

• DOI: 10.1067/mai.2000.107603
• 发表时间: 2000
• 期刊: The Journal of allergy and clinical immunology
• 作者: VanSambeek,R;Stevenson,DD;Baldasaro,M;Lam,BK;Zhao,J;Yoshida,S;Yandora,C;Drazen,JM;Penrose,JF
• 通讯作者: Penrose,JF