Purification and characterization of hepatic and intestinal phenol sulfotransferase with high affinity for benzo[a]pyrene phenols from channel catfish, Ictalurus punctatus.
Cytosol from channel catfish liver and intestinal mucosa has high sulfotransferase activity with low concentrations of 3-, 7-, or 9-hydroxybenzo[a]pyrene. To further investigate this conjugation pathway, sulfotransferase activity toward 9-hydroxybenzo[a]pyrene was isolated from catfish intestinal and hepatic cytosol by chromatography on anion exchange and PAP-agarose affinity columns. SDS-PAGE of the active fractions showed that one major band with molecular size of about 41,000 Da was isolated from intestine, while two bands of about 41,000 and 31,000 Da were obtained from liver. Antibodies against human phenol-sulfating sulfotransferase cross-reacted strongly with the 41,000-Da bands from liver and intestine, but weakly with the hepatic 31,000-Da protein. N-Terminal sequence information could not be obtained from the pure proteins. Following digestion, an internal sequence of 20 amino acid residues was obtained from the hepatic 41,000-Da protein, which matched a sequence found in several mammalian sulfotransferases. No fish sulfotransferase sequences were available for comparison. The identity of the hepatic 31,000-Da protein was not established. The purified 41,000-Da proteins had very high activities with 3-, 7-, or 9-hydroxybenzo[a]pyrene, with K(m) values in the 40-100 nM range and V(max) 125-300 nmol/min/mg of protein. Substrate inhibition was observed when the concentrations of hydroxylated benzo[a]pyrenes were above 0.5 microM. As well as benzo[a]pyrene phenols, the purified 41,000-Da sulfotransferases catalyzed sulfation of 2-naphthol, 4-nitrophenol, 4-methylumbelliferone, 7-(hydroxymethyl)-12-methylbenz[a]anthracene, dehydroepiandrosterone, estrone, and 17beta-estradiol. Phenolic compounds were the preferred substrates for the purified enzymes.
Tong
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Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610, USA.
James
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D000595:Amino Acid Sequence; D000818:Animals; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001564:Benzo(a)pyrene / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D002384:Catalysis / Q000187:drug effects; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006900:Hydroxylation; D007059:Ictaluridae; D007422:Intestines / Q000166:cytology / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010636:Phenols / Q000378:metabolism* / Q000494:pharmacology; D015203:Reproducibility of Results; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism
D010636:Phenols; D013431:Sulfates; D001564:Benzo(a)pyrene; D015239:Arylsulfotransferase