Abstract
Two active site-directed photoaffinity analogs, 5-[beta-32P]azido-UDP-glucuronic acid and 5-[beta-32P]azido-UDP-glucose, were used for the characterization of UDP-sugar-utilizing enzymes in human liver microsomes. Both compounds were recognized by human microsomal proteins: major photolabeled bands of 50-56 kDa were detected. Both photoincorporations were competitively decreased by increasing concentrations of either UDP-Glc or UDP-GlcUA, indicating a high affinity for both nucleotides. The patterns of photoaffinity labeling in the 50-56-kDa range by the two probes were significantly different, indicating the presence of different UDP-GlcUA- and UDP-Glc-specific enzymes of similar molecular mass. The presence of a UDP-Glc-dependent transferase was confirmed by the identification of an enzymatic activity catalyzing the formation of glucosides of the 6 alpha-hydroxylated bile acid hyodeoxycholic acid (3 alpha, 6 alpha-diOH (HDCA)) in the presence of UDP-Glc. The specific activity of 1.5-3.2 nmol/min/mg of protein was similar to that of 6 alpha-glucuronidation of HDCA. The apparent Km for UDP-Glc estimated with HDCA was 280 microM, and the formation of HDCA glucosides was strongly inhibited by UDP-GlcUA (apparent Ki = 7 microM). Evidence is presented that HDCA-specific UDP-glucuronosyltransferase (clone UGT2B4) expressed in V79 cells is not involved in glucosidation of HDCA and is not photolabeled with 5-[beta-32P]azido-UDP-Glc. Rigorous structure identification of the biosynthetic product proved that HDCA was glucosidated at the 6-position. Thus, this UDP-Glc-dependent activity catalyzing the biosynthesis of 6-O-glucosides of 6 alpha-hydroxylated bile acids represents a new pathway in the metabolism of these bile acids.
Highlights
A Novel UDP-Glc-specific Glucosyltransferase Catalyzing the Biosynthesis of 6-0-Glucosides of Bile Acids in Human Liver Microsomes*
Evidencewas presented that chenodeoxycholic probesweresignificantlydifferent,indicatingthe acid (3a,7a-diOH) and hyodeoxycholic acid (3a,6apresence of different UDP-GlcUA- and UDP-Glc-spe- diOH(HDCA)) were glucosidated i n vitro at
The presence postulated that, in addition to the glycosyltransferase reacof a UDP-Glc-dependent transferase wasconfirmed by tions involved in the biosynthesisof N-linked glycoproteins, the identification of an enzymatic activity catalyzing there exists in human liver microsomes a sugar nucleotidethe formationof glucosides of the 6a-hydroxylated bile independent glucosyltransferase that catalyzes the tranosfer acid hyodeoxycholic acid (3a,6a-diOH (HDCA))in the glucose from glucosyl donors to the 3-hydroxyl of bile acids presence of UDP-Glc
Summary
The separatedproteins were transferred cally, cloned UGT2B4 expressed in V79 cells is not involved from the gel to nitrocellulose, and Western blot analysis was perin the 6-0-glucosylation of HDCA This reaction is not carried out by the UDP-Glc-independent system described previously [11,14]. The reaction mixture (containing 10mg of microsomal protein, 0.15 mM [3H] HDCA (specific activity, -800 dpm/nmol), 2.5 mM UDP-Glc, 100 mM HEPES/NaOH (pH6.5), mM MgC12,and 5 mM saccharolactone inafinal volume of ml) was incubated a t 37 “C for 60 min. Ucts was accomplished by solid-phase extraction on Bond Elut Cla Incubation of Human Liver Microsomes with UDP-sugars cartridges (Varian-Analytichem), followedby preparative TLC, either in an underivatized form or as the methyl ester acetate derivatives, as previously described [10].’HNMR spectra were recorded in CDC13 with a Varian 300-MHz instrument and referenced to the CHC13 signal (7.26 ppm). The results on HDCA 6-0glucuronide formation areincluded in TableI1 for the purpose of comparison and as a check on the viability of the human
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