STRUCTURE AND REGULATION OF RAT ARYL SULFOTRANSFERASES

大鼠芳基磺基转移酶的结构和调控

• 批准号: 3297966
• 负责人: Charles N Falany
• 金额: $ 11.36万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297966
• 关键词: beta galactosidase; catecholamines; chemical carcinogenesis; complementary DNA; cytochrome P450; drug metabolism; enzyme mechanism; enzyme structure; gene expression; genetic regulation; genetic transcription; genetic translation; genome; glucuronosyltransferase; homeostasis; isozymes; messenger RNA; phenols; steroids; sulfation; sulfotransferase; tyrosine; western blottings

Sulfation is an important conjugation reaction involved in the modification of the biological activity and duration of action of many drugs and endogenous compounds such as steroids, catecholamines and bioactive peptides. Although sulfation of exogenous compounds is primarily a detoxification process, sulfation also has an important role in the activation of several compounds such as 4-aminoazobenzene and N-hydroxy-2-acetylamino- fluorene (N-OH-AAF) to ultimate carcinogens. The long-term goal of this project is to understand the role of sulfation in the metabolism of drugs and endogenous compounds as well as in the activation of chemical carcinogens. As observed for other drug metabolizing enzymes such as cytochromes P-450 and UDP- glucuronyltransferases, sulfotransferases represent a family of distinct isoenzymes. However, very little is known concerning the relationships, regulation or structure of these enzymes at the molecular level. The research proposed in this application is directed towards beginning the investigation of the molecular biology and transcriptional regulation of rat liver arylsulfotransferase IV (AST IV), an enzyme involved in the activation of N-OH-AAF to a potent carcinogen, the sulfation of amino-terminal tyrosines in small peptides, steroids and the metabolism of phenolic compounds add drugs. This project should also represent the first investigation into the molecular biology of a sulfotransferase. The goals of this project are three-fold. First, isolation and characterization of the AST IV cDNA will be accomplished. This will include hybrid-selection translation, expression of the enzymatically active protein in mammalian cells, raising an antibody to the beta-galactosidase-AST IV fusion protein and determination of the nucleotide sequence of the AST IV cDNA. Second, other sulfotransferase cDNAs related to AST IV will be isolated from the rat liver lambda gtll cDNA library using differential hybridization conditions to establish the number and relationships between the different rat liver aryl sulfotransferases. Third, the presence of AST IV mRNA will be localized and quantitated in various rat tissues to determine if the carcinogenic activity of compounds such as N-OH-AAF correlates with the distribution of AST IV and if significant levels of AST IV message are present in non-hepatic tissues where the enzyme may have a role in homeostasis. AST IV mRNA in the different tissues will be quantified by Northern blot techniques and the occurrence of AST IV message within specific tissues and cell types will be detected by in situ hybridization.

硫酸化是一个重要的共轭反应，涉及到 生物活性和作用持续时间的改变 许多药物和内源性化合物如类固醇， 儿茶酚胺和生物活性肽。 虽然硫酸化的 外源化合物主要是一种解毒过程， 硫酸化也在活化几种 化合物如4-氨基偶氮苯和N-羟基-2-乙酰氨基- 芴（N-OH-AAF）的最终致癌物。 远景目标 这个项目的目的是了解硫酸化在 药物和内源性化合物的代谢以及 化学致癌物的激活。 与其他药物相同 代谢酶，如细胞色素P-450和UDP- 葡萄糖醛酸转移酶，磺基转移酶代表一个家族， 不同的同工酶 然而，人们对它知之甚少。 这些酶的相互关系、调节或结构， 分子水平。 本申请中提出的研究针对 开始了分子生物学的研究， 大鼠肝脏芳基磺基转移酶IV的转录调控 (AST IV），一种参与N-OH-AAF活化为 潜在的致癌物质，氨基末端酪氨酸的硫酸化， 小肽、类固醇和酚类化合物的代谢 添加药物。 这个项目也应该是第一个 磺基转移酶的分子生物学研究。 该项目的目标有三个方面。 首先，隔离和 将完成AST IV cDNA的表征。 这 将包括杂交选择翻译， 哺乳动物细胞中的酶活性蛋白， β-半乳糖苷酶-AST IV融合蛋白的抗体，和 确定AST IV cDNA的核苷酸序列。 第二，与AST IV相关的其他磺基转移酶cDNA将被 从大鼠肝λ gt 11 cDNA文库中分离， 不同的杂交条件，以建立数量和 不同大鼠肝脂环素水平之间的关系 磺基转移酶 第三，AST IV mRNA的存在将是 定位和定量在不同的大鼠组织，以确定是否 化合物如N-OH-AAF的致癌活性与 与AST IV的分布相关，如果AST水平显著 IV信息存在于非肝组织中，其中酶可能 在体内平衡中发挥作用。 不同组织中AST IV mRNA 将通过北方印迹技术定量， 特定组织和细胞类型内的AST IV信息将被 通过原位杂交检测。