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Substrate specificity and characterization of rat liver p-nitrophenol, 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid UDP-glucuronosyltransferases.

ABSTRACT: Purified preparations of rat liver 17-hydroxysteroid, 3-hydroxyandrogen and p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferases were further characterized as to their substrate specificities, phospholipid-dependency and physical properties. The two steroid UDP-glucuronosyltransferases were shown to exhibit strict stereospecificity with respect to the conjugation of steroids and bile acids. These enzymes have been renamed 17 beta-hydroxysteroid and 3 alpha-hydroxysteroid UDP-glucuronosyltransferase to reflect this specificity for important endogenous substrates. An endogenous substrate has not yet been identified for the p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferase. The steroid UDP-glucuronosyltransferase activities were dependent on phospholipid for maximal catalytic activity. Complete delipidation rendered the UDP-glucuronosyltransferases inactive, and enzymic activity was not restored when phospholipid was added to the reaction mixture. After partial delipidation, phosphatidylcholine was the most efficient phospholipid for restoration of enzymic activity. Partial delipidation also altered the kinetic parameters of the 3 alpha-hydroxysteroid UDP-glucuronosyltransferase. The three purified UDP-glucuronosyltransferases are separate and distinct proteins, with different amino acid compositions and peptide maps generated by limited proteolysis with Staphylococcus aureus V8 proteinase. Some similarity was observed between the amino acid composition and limited proteolytic maps of the steroid UDP-glucuronosyltransferases, suggesting they are more closely related to each other than to the p-nitrophenol UDP-glucuronosyltransferase.

SUBMITTER: Falany CN

PROVIDER: S-EPMC1147098 | biostudies-other | 1986 Aug

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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Substrate specificity and characterization of rat liver p-nitrophenol, 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid UDP-glucuronosyltransferases.

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