The long-term goal of this research is to better understand the role that aryl sulfotransferase (AST) IV and alcohol sulfotransferase STa play in the cytotoxic, immunologic, mutagenic, and carcinogenic responses to drugs and other xenobiotics that either possess or are biotransformed into metabolites containing arylhydroxamic acid, N-hydroxy arylamine, or benzylic alcohol functional groups. These functional groups are usually encountered as intermediary metabolites of a multitude of different drugs and other xenobiotics containing arylamine, nitroaromatic, and arylamide functionalities as well as of xenobiotics possessing benzylic carbon atoms that are susceptible to metabolic oxidation. The research proposed in this application is based on the premise that quantitative analyses of the catalytic specificities and mechanisms of sulfotransferases and their intratissue localizations and distributions are essential for accurately predicting the potential for covalent alteration of macromolecules within individual cells following exposure to xenobiotics that possess these organic functional groups. Investigations on the relationships between AST IV and STa will focus on delineation of the degree of overlap in their specificities for phenols, benzylic alcohols, N-hydroxy arylamines, and arylhydroxamic acids as substrates and/ or inhibitors. Quantitative immunohistochemical methods will be used to determine overlap and/or complementarity in the intratissue localizations and distributions of the two enzymes in liver, skin, lung, and nasal mucosa of both male and female rats. In addition to these studies on the specificities and localizations of these two sulfotransferases, investigations on the catalytic mechanism of AST IV will be continued. These studies will incorporate active site-directed affinity labeling and peptide-sequencing in order to elucidate the structures of peptides at the two major regions of the active site (i.e., the PAPS-binding site and the binding site for a sulfuryl acceptor). Results from the affinity labeling experiments will then guide collaborative studies on site-directed mutagenesis to provide a more complete understanding of the catalytic mechanism of AST IV. Thus, the multifaceted approach utilized in this project will result in a significantly improved understanding of two of the major sulfotransferases involved in the formation of sulfuric acid esters that are implicated in the occurrence of chemical carcinogenesis and other toxic responses following exposure to numerous xenobiotics.
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