Sulfotransferases (ST) are Phase II metabolizing enzymes that sulfo- conjugate a variety of endogenous and exogenous compounds, such as biogenic amines (e.g., catecholamine), steroids (e.g., glucocorticoid steroids), drugs (e.g., propranolol and acetaminophen), and carcinogens (e.g., N-hydroxy-2-acetylaminofluorene and hydroxylated 2,3,7,8- tetrachlorodibenzo-p-dioxin). This proposal focuses on the isolation and characterization of the genes encoding for the three major types of sulfotransferases, i.g., alcohol/hydroxysteroid (ST-a), phenol/aryl(ST-p) and estrogen (ST-e) sulfotransferases. The underlying hypothesis is that cDNA cloning, expression and functional studies will offer unique insights into the biological mechanisms and physiological functions of STs and may permit more mechanistic insights into the molecular basis of sulfo-conjugation in man.
The specific aims of this research proposal are: 1) To clone, sequence and characterize the cDNAs encoding for the human alcohol/hydroxysteroid (STa), phenol/aryl (STp) and estrogen (STe) sulfotransferases. This will include a series of experiments aimed at screening and re-screening of a human male liver cDNA library using the polymerase chain reaction (PCR) cloned rat rST cDNA and the cloned human STs via cDNA expression in cultured cells. This will involve the expression of the cloned human STa STp and ST cDNAs in fibroblast cell lines, baculovirus system and Escherichia coli system and assaying for hST-a, hST-p and hST-e enzyme activities towards model pharmacological agents. 3) To establish mouse. This will involve experiments focusing on molecular cloning, characterization and expression of mouse STa, STp and STe cDNAs coupled with sulfo-conjugation studies of model pharmacological agents, and on the construction of an evolutionary phylogenetic tree to define the interrelationships of ST family of genes with respect to origin, enzymatic rates and specificities of each the ST gene. 4) To isolate, sequence and characterize the genomic DNA of the human STa, STp and STe genes to elucidate conserved domain(s) at the 5' flanking regions of the genes. This will involve screening of a human genomic library with their respective cloned human STa, STp and STe cDNAs and subsequent restriction-mapping and sequencing two kb 5'flanking regions of the genes. The results of the proposed experiments are likely to significantly enhance our fundamental understanding of the biological mechanisms for STs in the sulfate conjugation of drugs, xenobiotics and endobiotic.
