AMINO ACID CONJUGATION OF BILE ACIDS

胆汁酸与氨基酸甘氨酸和牛磺酸通过 肝脏，由胆汁酸辅酶A两种酶催化的代谢步骤 连接酶(BAL)和胆汁酸CoA：氨基酸N-酰基转移酶(BAT)。这个 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， 因此，确定胆汁酸酰胺化在胆汁淤积症中的作用。 胃肠道和肝胆系统。在现在 应用：(1)由于缺陷或BAL或BAT会产生 胆汁酸N-酰胺化缺陷，编码BAL的cDNA将是 从大鼠肝脏cDNA文库中分离、鉴定、测序和 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组织中相应的cDNAs 图书馆。(2)蝙蝠中表征其特性的氨基酸残基 氨基酸底物的选择(单独的牛磺酸或甘氨酸/ 牛磺酸)将被调查。确定关键氨基酸残基 对于BAT活性，推测的大鼠肝BAT基因(Kan-1)将是 表达以确定其底物特异性-如果它是牛磺酸- 其他rBAT基因将被克隆、测序和表达。 将在蝙蝠身上进行底物保护实验，以 确定胆汁的共价结合部位是哪种半胱氨酸 酸性辅酶A底物。LC-MS和MALDI飞行时间质谱学 将确定受保护的含有半胱氨酸的胰蛋白酶多肽 通过N-乙基马来酰亚胺的烷基化反应加成胆基辅酶A。其他 与BAT催化性能相关的氨基酸残基将 通过化学修饰和DNA位点特异性进行鉴定 新生实验。一种潜在的新陈代谢途径 甘氨酸结合胆汁酸将被探索。(3)淘汰赛模式 BAT缺乏症的研究将提供一种实验性的 用于测试丧失BAT活性的生理效应的系统。 Baat基因的组织将被确定，以便 129只带胎小鼠转化多能胚胎干细胞 正/负选择载体和识别包含蝙蝠的细胞 淘汰赛。

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.