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: 6235027
• 负责人: BING K LAM
• 金额: $ 16.22万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6235027
• 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（LT）合成酶是5-脂氧合酶的末端酶