STRUCTURE AND REGULATION OF RAT ARYL SULFOTRANSFERASES

硫化反应是一种重要的共轭反应，参与了 修改其生物活性和作用时间 许多药物和内源性化合物，如类固醇， 儿茶酚胺和生物活性多肽。尽管硫酸盐化 外源化合物主要是一个解毒过程， 硫酸盐化也在活化几种 4-氨基偶氮苯和N-羟基-2-乙酰氨基等化合物. 荧烯(N-OH-AAF)到最终致癌物。长期目标 这个项目的目的是了解硫酸盐化在环境中的作用 药物和内源性化合物的代谢以及在 化学致癌物的激活。与其他药物的观察结果相同 代谢酶如细胞色素P-450和UDP- 葡萄糖醛酸基转移酶，磺基转移酶代表一个家族 不同的同工酶。然而，人们对此知之甚少。 这些酶的关系、调节或结构 分子水平。 本申请中提出的研究针对的是 开始了分子生物学的研究和 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 强致癌物质，氨基末端酪氨酸的硫酸盐化 小肽、类固醇与酚类化合物的代谢 再加上毒品。这个项目也应该是第一个 磺基转移酶的分子生物学研究。 这个项目的目标有三个方面。第一，与世隔绝 将完成对AST IV基因的鉴定。这 将包括混合选择翻译、表达 在哺乳动物细胞中具有酶活性的蛋白，提高了 抗β-半乳糖苷酶-AST IV融合蛋白的抗体和 天冬氨酸氨基转移酶IV基因的核苷酸序列测定 第二，与AST IV相关的其他磺基转移酶cDNA将是 从大鼠肝脏lambda gtll文库中分离得到 不同杂交条件下建立的数量和 大鼠肝脏不同芳香基间的关系 磺基转移酶。第三，AST IV mRNA的存在将是 在大鼠不同组织中定位和定量，以确定是否 N-OH-AAF等化合物的致癌活性与 与AST IV的分布和AST显著水平的关系 IV信息存在于非肝脏组织中，其中酶可能 在动态平衡中发挥作用。不同组织中AST IV基因的表达 将通过Northern印迹技术进行量化，并发生 在特定组织和细胞类型内的AST IV消息将是 通过原位杂交检测。

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.