title: Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide.
abstract: 1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency. 2. Addition of Mg2+(to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. 3. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. 4. These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.
authors.affiliation: a United States Environmental Protection Agency (US EPA), ORD, NHEERL, Mid-Continent Ecology Division , Duluth , MN , USA.
authors.forename: Melanie A
authors.initials: MA
authors.lastname: Ladd
authors.affiliation: a United States Environmental Protection Agency (US EPA), ORD, NHEERL, Mid-Continent Ecology Division , Duluth , MN , USA.
authors.forename: Patrick N
authors.initials: PN
authors.lastname: Fitzsimmons
authors.affiliation: a United States Environmental Protection Agency (US EPA), ORD, NHEERL, Mid-Continent Ecology Division , Duluth , MN , USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Nichols
mesh_terms: D000408:Alamethicin / Q000494:pharmacology*; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology*; D008099:Liver; D008110:Liver Extracts / Q000378:metabolism*; D014337:Trout
chemical_list: D007476:Ionophores; D008110:Liver Extracts; D000408:Alamethicin; D014453:Glucuronosyltransferase
keywords: Alamethicin; UDP-glucuronosyltransferase; UGT; glucuronidation; liver S9 fraction; rainbow trout