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 sulfotran sferase activity with low concentrations of 3-, 7-, or 9-hydroxybenzo[a]pyr ene. To further investigate this conjugation pathway, sulfotransferase acti vity toward 9-hydroxybenzo[a]pyrene was isolated from catfish intestinal an d hepatic cytosol by chromatography on anion exchange and PAP-agarose affin ity columns. SDS-PAGE of the active fractions showed that one major band wi th 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 a gainst human phenol-sulfating sulfotransferase cross-reacted strongly with the 41,000-Da bands from liver and intestine, but weakly with the hepatic 3 1,000-Da protein. N-Terminal sequence information could not be obtained fro m the pure proteins, Following digestion, an internal sequence of 20 amino acid residues was obtained from the hepatic 41,000-Da protein, which matche d a sequence found in several mammalian. sulfotransferases, No fish sulfotr ansferase sequences were available for comparison. The identity of the hepa tic 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. Sub strate inhibition was observed when the concentrations of hydroxylated benz o[a]pyrenes were above 0.5 mu M. As well as benzo[a]pyrene phenols, the pur ified 41,000-Da sulfotransferases catalyzed sulfation of 2-naphthol, 4-nitr ophenol, 4-methylumbelliferone, 7-(hydroxymethyl)-12-methylbenz[a]anthracen e, dehydroepiandrosterone, estrone, and 17 beta-estradiol, Phenolic compoun ds were the preferred substrates for the purified enzymes. (C) 2000 Academi c Press.