ARYL AND ALCOHOL SULFOTRANSFERASES IN DRUG METABOLISM

这项研究的长期目标是更好地了解和更多 可靠地预测芳基磺基转移酶（AST）IV和酒精 磺基转移酶STa在细胞毒性、致突变和/或致癌中发挥作用 对药物、致癌物和其他外源性物质的反应。 准确 预测涉及磺基转移酶的代谢反应， 特别重要的是，对于那些拥有或被转化为 转化为含有苄醇、芳基异羟肟酸和N- 羟基芳基胺官能团，因为这些官能团硫酸化 基团通常是形成化学反应性的关键步骤， 能够与细胞形成共价键的代谢物 大分子这种与关键细胞分子的共价键合是 初始步骤导致多种细胞毒性的，免疫的， 致突变和致癌反应。这项研究提出， 应用的前提是， 磺基转移酶的催化特异性和机制，以及 这些酶在特定细胞内的定位和调节， 组织如肝脏，对于准确预测 潜在的共价改变细胞内的大分子 单个细胞。 AST IV的定量构效关系研究 和STa将集中在疏水和空间特性 与这些酶结合和催化所必需的。这些研究将 还提供了普遍适用的定量比较， 这两种磺基转移酶的立体选择性通过使用同源的 一系列手性底物。该项目的第二个具体目标是 鉴定和定位特定的氨基酸残基， AST IV和STa的底物结合和催化活性。这 研究继续成功地使用核糖核苷酸 二醛亲和标记试剂，用于阐明 3 '-磷酸腺苷5'-磷酸硫酸盐的结合位点，并采用新的 测定硫酰受体上残留物的方法 绝佳的价钱 这些新方法将利用一系列同源的底物- 含有溴乙酰氨基基团的类似物作为AST IV的亲和标记。 这些亲和标记被设计为提供关于相对生物的信息。 特定氨基酸残基与硫酰基位点的距离 转移到酶上。这项研究的第三个主要组成部分是一个 探索AST IV和ST a异质性的基础， 原位杂交技术检测肝细胞跨肝小叶的跨膜表达 免疫组织化学分析。这些调查将提供 关于这两种类型的关系的重要新信息 肝脏内的磺基转移酶。

The long-term objective of this research is to better understand and more reliably predict the roles that aryl sulfotransferase (AST) IV and alcohol sulfotransferase STa play in the cytotoxic, mutagenic and/or carcinogenic responses to drugs, carcinogens, and other xenobiotics. The accurate prediction of metabolic reactions involving sulfotransferases is particularly important for molecules that either possess or are converted into metabolites containing benzylic alcohol, arylhydroxamic acid, and N- hydroxy arylamine functional groups, since sulfation of these functional groups is often the critical step in the formation of chemically reactive metabolites that are capable of forming covalent bonds with cellular macromolecules. Such Covalent bonding with critical cellular molecules is the initial step leading to a variety of cytotoxic, immunologic, mutagenic, and carcinogenic responses. The research proposed in this application is based on the premise that quantitative analyses of the catalytic specificities and mechanisms of sulfotransferases, as well as the localizations and regulation of these enzymes within specific cells in tissues such as the liver, are essential for accurately predicting the potential for covalent alteration of cellular macromolecules within individual cells. Investigations on quantitative structure-activity relationships for AST IV and STa will be focused on the hydrophobic and steric characteristics required for binding and catalysis with these enzymes. These studies will also provide generally applicable quantitative comparisons of the stereoselectivity of the two sulfotransferases through use of a homologous series of chiral substrates. A second specific aim of this project is to identify and locate specific amino acid residues that contribute to the substrate binding and catalytic activity of AST IV and STa. This investigation both continues the successful use of ribonucleotide dialdehyde affinity labeling reagents for elucidation of residues at the binding site for 3'-phosphoadenosine 5'-phosphosulfate and employs new approaches for the determination of residues at the sulfuryl acceptor site. These new methods will utilize a homologous series of substrate- analogs containing bromoacetamido groups as affinity labels for AST IV. These affinity labels are designed to provide information on the relative distances of specific amino acid residues from the site of sulfuryl transfer on the enzyme. The third major component of this research is an exploration of the basis for heterogeneity of AST IV and STa within hepatocytes across the rat liver lobule by combining in situ hybridization with immunohistochemical analyses. These investigations will provide important new information on the relation of these two types of sulfotransferases within liver.