AMINO ACID CONJUGATION OF BILE ACIDS

胆汁酸的氨基酸缀合

• 批准号: 2752270
• 负责人: STEPHEN BARNES
• 金额: $ 18.49万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-15 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2752270
• 关键词: acid thiol ligase; active sites; acyltransferase; chemical conjugate; cholanate compound; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; gene targeting; genetic library; genetically modified animals; glycine; high performance liquid chromatography; laboratory mouse; laboratory rabbit; laboratory rat; liver metabolism; matrix assisted laser desorption ionization; protein sequence; steroid metabolism; taurine

Bile acids are conjugated with the amino acids glycine and taurine by the liver, a metabolic step catalyzed by two enzymes, bile acid CoA ligase (BAL) and bile acid CoA: amino acid N-acyltransferase (BAT). The long term goal of this project is to use molecular biology approaches to establish the physiological importance of amidation of bile acids, and hence to determine the role of bile acid amidation in diseases of the gastrointestinal and hepatobiliary systems. In the present application: (1) Since a deficiency or either BAL or BAT will create a defect in bile acid N-acyl amidation, cDNAs encoding BAL will be isolated from a rat liver cDNA library, characterized, sequenced and expressed in a suitable expression system. cDNAs encoding rat BAL will be used to isolate the corresponding cDNAs in human and mouse liver cDNA libraries. (2) The amino acid residues in BAT that characterize its choice of amino acid substrates (either taurine alone, or glycine/ taurine) will be investigated. To identify critical amino acid residues for BAT activity, a putative rat liver BAT cDNA (Kan-1) will be expressed to determine its substrate specificity- if it is a taurine- specific BAT, other rBAT cDNAs will be cloned, sequenced and expressed. Substrate protection experiments will be carried out on BATs to determine which cysteine is the site of covalent attachment of the bile acid CoA substrate. HPLC-MS and MALDI-time-of-flight mass spectrometry will identify the cysteine-containing tryptic peptide that is protected by addition of cholyl CoA from alkylation by N-ethylmaleimide. Other amino acid residues associated with the catalytic properties of BAT will be identified by chemical modification, and by DNA site-specific metagenesis experiments. A potential new pathway of metabolism of glycine-conjugated bile acids will be explored. (3) A knockout model of BAT deficiency will be developed so as to provide an experimental system for testing the physiological effects of loss of BAT activity. The organization of the Baat gene will be determined in order to transform pluripotent embryonic stem cells from the 129 mice with a Baat positive/negative selection vector and identify cells containing the BAT knockout.

胆汁酸与氨基酸甘氨酸和牛磺酸通过 肝脏，由两种酶催化的代谢步骤，胆汁酸CoA 连接酶（BAL）和胆汁酸CoA：氨基酸N-酰基转移酶（BAT）。 的 这个项目的长期目标是使用分子生物学方法 为了确定胆汁酸酰胺化的生理重要性， 从而确定胆汁酸酰胺化在疾病中的作用， 胃肠和肝胆系统。 本 应用：（1）由于BAL或BAT的不足将导致 胆汁酸N-酰基酰胺化缺陷，编码BAL的cDNA将被 从大鼠肝脏cDNA文库中分离，表征，测序， 在合适的表达系统中表达。 编码大鼠BAL的cDNA将 用于分离人和小鼠肝cDNA中相应的cDNA 图书馆. (2)BAT中表征其生物学特性的氨基酸残基 氨基酸底物的选择（单独的牛磺酸或甘氨酸/ 牛磺酸）进行研究。 鉴定关键氨基酸残基 对于BAT活性，将从大鼠肝脏中提取一个假定的BAT cDNA（Kan-1）， 表达以确定其底物特异性-如果是牛磺酸- 除了特异性BAT外，其他rBAT cDNA将被克隆、测序和表达。 将在BAT上进行基底保护实验， 确定哪一个半胱氨酸是胆汁的共价连接位点 酸性CoA底物。 HPLC-MS和MALDI-飞行时间质谱 将鉴定受保护的含半胱氨酸的胰蛋白酶肽 通过添加来自N-乙基马来酰亚胺烷基化的胆酰CoA。 其他 与BAT的催化特性相关的氨基酸残基将 通过化学修饰和DNA位点特异性 后生实验 一种潜在的新的代谢途径 将探索甘氨酸结合的胆汁酸。 (3)一个出色的模特 将制定最佳可得技术缺陷的评估办法， 用于测试BAT活性丧失的生理效应的系统。 Baat基因的组织将被确定，以便 用Baat转化129只小鼠的多能胚胎干细胞， 阳性/阴性选择载体，并鉴定含有BAT的细胞 漂亮