HUMAN AMINO ACID CONJUGATION OF BILE ACIDS

人氨基酸与胆汁酸的缀合

• 批准号: 2145590
• 负责人: STEPHEN BARNES
• 金额: $ 15.49万
• 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 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145590
• 关键词: acid thiol ligase; acyltransferase; bile; chemical conjugate; cholanate compound; enzyme activity; enzyme mechanism; enzyme structure; genetic mapping; human tissue; isozymes; laboratory rabbit; laboratory rat; liver metabolism; protein purification; protein sequence; steroid metabolism

The long term goals of this project are to understand the biochemistry, molecular biology and cell biology in humans of the two enzymes, bile acid CoA synthetase (hBAS) and bile acid CoA: amino acid N-acyltransferase (hBAT), which are responsible for the biosynthesis of the amino acid conjugates of bile acids. The naturally occurring C24 bile acids in human bile are found almost exclusively as their amino acid conjugates. These bile acid conjugates are by far the principal solutes of human bile and their secretion is the major determinant of bile flow. In bile and the small intestine, bile acid amino acid conjugates form mixed micellar complexes with water-insoluble lipid species such as phospholipids, monoacylglycerols, free fatty acids and cholesterol, thereby performing important physiological functions both in normal health and disease. To fully understand how hBAS and hBAT are involved in the synthesis of these conjugates in humans requires knowledge of the chemistry of these enzymes (their primary amino acid sequence), their genetic organization (nucleotide sequence of cDNAs encoding them and the gene(s) from which they were derived and their chromosomal localization), and the regions of each enzyme which are responsible for their catalytic properties. The specific aims of this application are (1) to characterize the chemical and biochemical properties of purified hBAT by determining whether it has Ping-Pong Bi Bi reaction mechanism, evaluating the importance of its three cysteine residues (cys-235, cys-372 and cys-373) to enzyme function, and examining the rules which govern its substrate specificity; (2) to purify hBAS, characterize its biochemical and chemical properties (as for hBAT) and raise specific polyclonal anti-hBAS antibodies in rabbits; (3) to use the hBAT cDNA to determine the heterogeneity of RNA sequences related to hBAT and the chromosomal localization of the hBAT gene. When these specific aims have been accomplished, it will be possible in future granting periods to clone hBAS and to further analyze which regions of the hBAS and hBAT are essential for enzyme catalytic function and substrate specificity.

这个项目的长期目标是了解生物化学， 分子生物学和细胞生物学中人体的两种酶，胆汁酸 CoA合成酶（hBAS）和胆汁酸CoA：氨基酸N-酰基转移酶 （hBAT），其负责氨基酸的生物合成 胆汁酸的结合物。人体内天然存在的C24胆汁酸 胆汁几乎完全以其氨基酸缀合物的形式存在。这些 胆汁酸缀合物是人胆汁的主要溶质， 它们的分泌是胆汁流动的主要决定因素。在胆汁和 小肠，胆汁酸氨基酸结合物形成混合胶束 与水不溶性脂质物质如磷脂的复合物， 单酰基甘油、游离脂肪酸和胆固醇，从而执行 在正常健康和疾病中的重要生理功能。到 充分了解hBAS和hBAT是如何参与这些合成的 在人体中使用这些酶的化学知识 （它们的主要氨基酸序列），它们的遗传组织 （编码它们的cDNA的核苷酸序列和编码它们的基因） 它们的来源和它们的染色体定位），以及 每一种酶都负责它们的催化特性。的 本申请的具体目的是（1）表征化学品， 通过测定纯化的hBAT是否具有 乒乓毕毕反应机理，评价其三个重要性 半胱氨酸残基（cys-235、cys-372和cys-373）对酶功能的影响，和 研究控制其底物特异性的规则;（2）纯化 hBAS，表征其生化和化学特性（与hBAT相同） 并在家兔体内制备特异性多克隆抗hBAS抗体; hBAT cDNA来确定与以下相关的RNA序列的异质性： hBAT和hBAT基因的染色体定位。当这些 具体目标已经实现，将来有可能 给予克隆hBAS的时间，并进一步分析 hBAS和hBAT是酶催化功能和底物所必需 的特异性