655 research outputs found

    Heterologous Systems for Expression of Mammalian Sulfotransferases

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    This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases. In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodie

    Localization of Minoxidil Sulfotransferase in Rat Liver and the Outer Root Sheath of Anagen Pelage and Vibrissa Follicles

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    The precise biochemical mechanism and site(s) of action by which minoxidil stimulates hair growth are not yet clear. Minoxidil sulfate is the active metabolite of minoxidil, with regard to smooth muscle vasodilation and hair growth. Formation of minoxidil sulfate is catalyzed by specific PAPS-dependent sulfotransferase(s) and minoxidil-sulfating activities have been previously reported to be present in liver and hair follicles. One of these minoxidil-sulfating enzymes has been purified from rat liver (rat minoxidil sulfotransferase, MST) and a rabbit anti-MST antibody has been prepared. Using this anti-MST antibody, we have immunohistochernically localized minoxidil sulfotransferase in the liver and anagen hair follicles from rat. In rat pelage and vibrissa follicles, this enzyme is localized within the cytoplasm of epithelial cells in the lower outer root sheath. Although the immunolocalization of MST might not necessarily correlate with the MST activity known to be present in anagen follicles, the results of this study strongly suggest that the lower outer root sheath of the hair follicle may serve as a site for the sulfation of topically applied minoxidil

    In Vitro Inhibition of Human Hepatic and cDNA-Expressed Sulfotransferase Activity with 3-Hydroxybenzo[a]pyrene by Polychlorobiphenylols

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    Sulfonation is a major phase II biotransformation reaction. In this study, we found that several polychlorobiphenylols (OH-PCBs) inhibited the sulfonation of 3-hydroxybenzo[a]pyrene (3-OH-BaP) by human liver cytosol and some cDNA-expressed sulfotransferases. At concentrations > 0.15 μM, 3-OH-BaP inhibited its own sulfonation in cytosol fractions that were genotyped for SULT1A1 variants, as well as with expressed SULT1A1*1, SULT1A1*2, and SULT1E1, but not with SULT1A3 or SULT1B1. The inhibition fit a two-substrate kinetic model. We examined the effects of OH-PCBs on the sulfonation of 0.1 or 1.0 μM 3-OH-BaP, noninhibitory and inhibitory substrate concentrations, respectively. At the lower 3-OH-BaP concentration, OH-PCBs with a 3-chloro-4-hydroxy substitution pattern were more potent inhibitors of cytosolic sulfotransferase activity [with concentrations that produced 50% inhibition (IC(50)) between 0.33 and 1.1 μM] than were OH-PCBs with a 3,5-dichloro-4-hydroxy substitution pattern, which had IC(50) values from 1.3 to 6.7 μM. We found similar results with expressed SULT1A1*1 and SULT1A1*2. The OH-PCBs were considerably less potent inhibitors when assay tubes contained 1.0 μM 3-OH-BaP. The inhibition mechanism was noncompetitive, and our results suggested that the OH-PCBs competed with 3-OH-BaP at an inhibitory site on the enzyme. The OH-PCBs tested inhibited sulfonation of 3-OH-BaP by SULT1E1, but the order of inhibitory potency was different than for SULT1A1. SULT1E1 inhibitory potency correlated with the dihedral angle of the OH-PCBs. The OH-PCBs tested were generally poor inhibitors of SULT1A3- and SULT1B1-dependent activity with 3-OH-BaP. These findings demonstrate an interaction between potentially toxic hydroxylated metabolites of PCBs and polycyclic aromatic hydrocarbons, which could result in reduced clearance by sulfonation

    DIFFERENTIAL REGULATION OF INDIVIDUAL SULFOTRANSFERASE ISOFORMS BY PHENOBARBITAL IN MALE RAT LIVER

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    This paper is available online at http://www.dmd.org ABSTRACT: Xenobiotics that induce the cytochromes P450 also produce changes in rat hepatic sulfotransferase (SULT) gene expression. In the present study, male Sprague-Dawley rats were treated for 3 consecutive days with doses of phenobarbital (PB) that induce cytochrome P450 2B1/2 expression. The effects of PB treatment on hepatic aryl SULT (SULT1) and hydroxysteroid SULT (SULT2) mRNA and immunoreactive protein levels and on mRNA expression of individual SULT1 and SULT2 enzyme isoforms were characterized. PB suppressed SULT1A1 mRNA levels, increased the expression of the SULT-Dopa/tyrosine isoform, and did not produce significant changes in SULT1C1 and SULT1E2 mRNA expression. In rats injected with the highest test dose of PB (100 mg/kg), hepatic SULT1A1 mRNA levels were decreased to ϳ42% of control levels and SULT-Dopa/tyrosine mRNA levels were increased to ϳ417% of vehicle-treated control levels. Like the SULT1 subfamily, individual members of the SULT2 gene subfamily were differentially affected by PB treatment. PB (35, 80, and 100 mg/kg) suppressed SULT20/21 mRNA expression to ϳ61, ϳ30, and ϳ41% of vehicle-treated control levels, respectively. In contrast, SULT60 mRNA levels were increased to ϳ162% of control levels and SULT40/41 mRNA levels were increased to ϳ416% of vehicletreated control levels in rats treated with 100 mg/kg PB. These studies support a complex role for PB-mediated effects on the SULT multigene family in rat liver. Because individual SULT1 and SULT2 enzyme isoforms are known to metabolize a variety of potentially toxic substrates, varied responses to PB among members of the SULT multigene family might have important implications for xenobiotic hepatotoxicity

    Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium radiobacter AD1

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    The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the α/β-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 → Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.

    Structural and Chemical Profiling of the Human Cytosolic Sulfotransferases

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    The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique “chemical fingerprints” for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural “priming” of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone

    Garlic Extract Diallyl Sulfide (DAS) Activates Nuclear Receptor CAR to Induce the Sult1e1 Gene in Mouse Liver

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    Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car+/+ and Car−/− mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,200 fold) and protein in the livers of Car+/+ females but not of Car−/− female mice. DAS also induced other CAR-regulated genes such as Cyp2b10, Cyp3a11 and Gadd45β. Compared with the rapid increase of these mRNA levels, which began as early as 6 hourrs after DAS treatment, the levels of SULT1E1 mRNA began increasing after 24 hours. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estrone sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice
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