BIOCHEMICAL AND GENOMIC CHARACTERIZATION OF HUMAN LEUKOTRIENE C4 SYNTHASE

人白三烯 C4 合成酶的生物化学和基因组特征

• 批准号: 6099538
• 负责人: BING K LAM
• 金额: $ 16.87万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6099538
• 关键词: affinity chromatography; asthma; chemical kinetics; enzyme activity; epoxides; fatty acid biosynthesis; genetic regulation; glutathione; human tissue; immunocytochemistry; leukotrienes; lipoxygenase; lung; molecular cloning; mutant; plasmids; protein structure function; recombinant proteins; site directed mutagenesis

Leukotriene (LT) synthase is the terminal enzyme of 5-lipoxygenase pathway that is involved in conjugating the unstable epoxide intermediate, LTA4, with glutathione (GSH) to form LTC4. Hence, LTC4 synthase can regulate the biosynthesis of this potent mediator, which contributes to the pathobiology of bronchial asthma through its metabolites, LTD4 and LTE4. The overall objective of this project is to define tissue/cellular and subcellular distribution of LTC4 synthase, to characterize in biochemical and molecular terms the conjugation of LTA4 with reduced GSH by this novel protein, and to clone the gene so as to study its regulation. The cellular localization of LTC4 synthase in human lung tissue from individuals with and without asthma as well as the subcellular distribution of LTC4 synthase in normal and leukemic cell lines will be studied by immunohistochemistry and by immunogold labeling electron microscopy with polyclonal antibodies raised against purified LTC4 synthase derived from human lung. The kinetic studies will be performed on recombinant enzyme purified from baculovirus-infected insect cells after S-hexyl GSH chromatography and LTC affinity chromatography or if necessary, by antibody affinity chromatography or affinity chromatography of a tagged protein with subsequent removal of the tag. The structural relationship of LTC4 synthase to function will be determined by examining the enzyme kinetics of the mutated protein obtained by site-directed mutagenesis and by the use of substrate analogs. The LTC4 synthase gene will be cloned from a human leukocyte EMBL-3 genomic library using near full length LTC4 synthase cDNA as a probe. Up to 3 kilobases (kb) of the 5 prime flanking region of the gene will be sequenced and its cis-acting element will be examined with a DNase I hypersensitivity assay and by transfection of enhancerless and promoterless human growth hormone expression plasmid (p-phi-GH).

白三烯合酶是5-脂氧合酶的末端酶 与不稳定的环氧化物结合有关的途径 将中间体LTA 4与谷胱甘肽（GSH）反应以形成LTC 4。因此，LTC 4 合成酶可以调节这种有效介质的生物合成， 有助于支气管哮喘的病理生物学，通过其 代谢物LTD 4和LTE 4。该项目的总体目标是 定义LTC 4合酶的组织/细胞和亚细胞分布， 在生物化学和分子方面表征LTA 4的缀合 通过这种新的蛋白质减少GSH，并克隆该基因， 研究它的规则。LTC 4合酶在大肠杆菌中的细胞定位 来自患有和没有哮喘的个体的人肺组织以及 正常和白血病细胞中LTC 4合酶亚细胞分布 将通过免疫组织化学和免疫金标记研究细胞系 电子显微镜下用多克隆抗体对纯化的 来源于人肺的LTC 4合酶。动力学研究将是 对从杆状病毒感染的昆虫中纯化的重组酶进行 S-己基GSH层析和LTC亲和层析后的细胞 或如果需要，通过抗体亲和层析或亲和层析， 标记的蛋白质的层析，随后除去标记。 LTC 4合酶的结构与功能的关系将是 通过检查突变蛋白的酶动力学来确定 通过定点诱变和使用底物获得 类似物LTC 4合酶基因将从人白细胞中克隆 使用近全长LTC 4合酶cDNA作为EMBL-3基因组文库 探针该基因5 ′侧翼区的最多3个内切酶（kb） 将被测序，其顺式作用元件将被检查， DNA酶I超敏性测定和通过转染无增强子和 无启动子的人生长激素表达质粒（p-phi-GH）。