Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT).

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.

Photoaffinity labeling studies of the human recombinant UDP-glucuronosyltransferase, UGT1*6, with 5-azido-UDP-glucuronic acid.

Recombinant human liver UDP-glucuronosyltransferase (UGT), UGT1*6, which catalyzes the glucuronidation of small phenols, previously expressed in a V79 cell line (1) was photolabeled with [beta-32P]5N3UDP-glucuronic acid ([beta-32P]5N3UDP-GlcUA). Two polypeptides with an approximate molecular weight of 54 kDa were extensively photolabeled in the recombinant cell line while the nontransfected cell line showed no photoincorporation in this area. The identity of the two polypeptides as UGTs, which correspond to two different glycosylation forms of the same enzyme, was confirmed by Western blot using a polyclonal monospecific antibody directed against the 120 amino acids of the N-terminal end of UGT1*6.

Arginine 52 and histidine 54 located in a conserved amino-terminal hydrophobic region (LX2-R52-G-H54-X3-V-L) are important amino acids for the functional and structural integrity of the human liver UDP-glucuronosyltransferase UGT1*6.

The hepatic UDP-glucuronosyltransferase UGT1*6 is actively involved in the glucuronidation of short and planar phenols in humans. Based on the irreversible inhibition of the enzyme on chemical modification by 2,3-butanedione and diethyl pyrocarbonate, the roles of His54 and Arg52 were investigated by oligonucleotide site-directed mutagenesis. These amino acids belong to a consensus sequence LX2-R52-G-H54-X3-V-L located in a conserved hydrophobic region of the variable amino-terminal domain of UGT.

Application of photoaffinity labeling with [11,12-3H]all-trans-retinoic acid to characterization of rat liver microsomal UDP-glucuronosyltransferase(s) with activity toward retinoic acid.

[3H]All-trans-retinoic acid has been shown to be an effective photoaffinity label for microsomal UDP-glucuronosyltransferases. Labeling of rat liver microsomal proteins with [3H]all-trans-retinoic acid and [32P]5-azido-UDP-glucuronic acid has shown that at least one protein in the 50-56 kDa mass range encompassing the UDP-glucuronosyltransferases photoincorporated both probes. The fraction of solubilized microsomal protein eluted from a UDP-hexanolamine affinity column with 50 microM UDP-glucuronic acid contained two protein bands, both of which photoincorporated [3H] all-trans-retinoic acid and were detected on Western blot by anti-UDP-glucuronosyltransferase antibodies.

Glucuronidation of all-trans-retinoic acid and 5,6-epoxy-all-trans-retinoic acid. Activation of rat liver microsomal UDP-glucuronosyltransferase activity by alamethicin.

The effects of detergent, alamethicin (a channel-forming peptide), and the inducers phenobarbital and 3-methylcholanthrene on glucuronidation of all-trans-retinoic acid (atRA) and 5,6-epoxy-atRA have been investigated using liver microsomes from Sprague-Dawley and Fischer 344 rats. Conditions for enzymatic glucuronidation were optimized for substrate concentration, protein, and time by using atRA and Sprague-Dawley microsomes. With detergent-activated Sprague-Dawley microsomes, 5,6-epoxy-atRA was shown to be a significantly better substrate than atRA for microsomal glucuronidation (263 vs.