STRUCTURE AND REGULATION OF RAT ARYL SULFOTRANSFERASES

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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.