BIOCHEMICAL AND GENOMIC CHARACTERIZATION OF HUMAN LEUKOTRIENE C4 SYNTHASE

白三烯(LT)合成酶是5-脂氧合酶的末端酶 参与不稳定环氧化物结合的途径 中间体LTA4与谷胱甘肽(GSH)形成LTC4。因此，LTC4 合成酶可以调节这种强有力的介质的生物合成，这是 通过其在支气管哮喘病理生物学中的作用 代谢物LTD4和LTE4。这个项目的总体目标是 定义LTC4合酶的组织/细胞和亚细胞分布，以 LTA4偶联反应的生化和分子表征 并克隆该基因，以期通过该新蛋白降低GSH。 研究它的规则。LTC4合酶在大鼠脑内的细胞定位 来自哮喘患者和非哮喘患者的人肺组织以及 LTC4合酶在正常和白血病细胞中的亚细胞分布 将通过免疫组织化学和免疫金标记法研究这些株系。 抗纯化多克隆抗体的电子显微镜观察 来源于人肺的LTC4合成酶。动力学研究将是 从杆状病毒感染昆虫中纯化重组酶的研究 S-己基GSH层析和LTC亲和层析后的细胞 或如有必要，通过抗体亲和层析或亲和 标记蛋白质的层析，随后去除标记。 LTC4合成酶与功能的结构关系将是 通过检测突变蛋白质的酶动力学来确定 通过定点突变和使用底物获得 类比。LTC4合成酶基因将从人类白细胞中克隆出来 以近全长LTC4合成酶基因为模板的Embl-3基因组文库 探测器。该基因5个碱基侧翼区最高可达3kb 将被测序，其顺式作用元件将用 脱氧核糖核酸酶I超敏试验及无增强子和 无启动子人生长激素表达载体(p-Phi-GH)。

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).