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Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa.

ABSTRACT: The aim of this work was to determine if a non-mammalian species had multiple UDP-glucuronosyltransferase (UDPGT) isoforms. At least six highly purified UDPGT isoenzymes were partially resolved by anion-exchange chromatography and UDP-hexanolamine-Sepharose 4B affinity chromatography from liver microsomes of a fish, the plaice. Q-Sepharose FF, chromatofocusing and affinity-chromatographic procedures were employed to separate and purify the phenol UDPGT isoform to apparent homogeneity. The purified enzyme conjugated 1-naphthol, but not bilirubin or steroids, and displayed a pI of 7.0 and a subunit molecular mass of 55 kDa. Bilirubin and testosterone UDPGT activities were more labile and, although purified over 200-fold, these preparations also contained the phenol UDPGT and had multiple polypeptides with molecular masses of 52-57 kDa. Antisera to rat bilirubin/phenol UDPGT and testosterone/phenol UDPGT isoforms cross-reacted strongly with the partially purified plaice UDPGT isoforms of molecular masses 52, 53 and 57 kDa and less strongly with phenol UDPGT 54 kDa and 56 kDa isoforms. Fish and mammalian UDPGTs therefore apparently possess a high degree of evolutionary conservation.

SUBMITTER: Clarke DJ

PROVIDER: S-EPMC1132655 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

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Project description:2-Amino-9H-pyrido[2,3-b]indole (A?C) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAA) that arise during the burning of tobacco and cooking of meats. UDP-glucuronosyltransferases (UGT) detoxicate many procarcinogens and their metabolites. The genotoxic N-hydroxylated metabolite of A?C, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-A?C), undergoes glucuronidation to form the isomeric glucuronide (Gluc) conjugates N(2)-(?-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HON(2)-Gluc) and O-(?-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HN(2)-O-Gluc). A?C-HON(2)-Gluc is a stable metabolite but A?C-HN(2)-O-Gluc is a biologically reactive intermediate, which covalently adducts to DNA at levels that are 20-fold higher than HONH-A?C. We measured the rates of formation of A?C-HON(2)-Gluc and A?C-HN(2)-O-Gluc in human organs: highest activity occurred with liver and kidney microsomes, and lesser activity was found with colon and rectum microsomes. A?C-HN(2)-O-Gluc formation was largely diminished in liver and kidney microsomes, by niflumic acid, a selective inhibitor UGT1A9. In contrast, A?C-HON(2)-Gluc formation was less affected and other UGT contribute to N(2)-glucuronidation of HONH-A?C. UGT were reported to catalyze the formation of isomeric Gluc conjugates at the N(2) and N3 atoms of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), the genotoxic metabolite of PhIP. However, we found that the N3-Gluc of HONH-PhIP also covalently bound to DNA at higher levels than HONH-PhIP. The product ion spectra of this Gluc conjugate acquired by ion trap mass spectrometry revealed that the Gluc moiety was linked to the oxygen atom of HONH-PhIP and not the N3 imidazole atom of the oxime tautomer of HONH-PhIP as was originally proposed. UGT1A9, an abundant UGT isoform expressed in human liver and kidney, preferentially forms the O-linked Gluc conjugates of HONH-A?C and HONH-PhIP as opposed to their detoxicated N(2)-Gluc isomers. The regioselective O-glucuronidation of HONH-A?C and HONH-PhIP, by UGT1A9, is a mechanism of bioactivation of these ubiquitous HAAs.

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Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa.

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