A novel UDP-Glc-specific glucosyltransferase catalyzing the biosynthesis of 6-O-glucosides of bile acids in human liver microsomes

• Published in: The Journal of biological chemistry Vol. 268; no. 20; pp. 15127 - 15135
• Main Authors: Radominska, A, Little, J, Pyrek, J S, Drake, R R, Igari, Y, Fournel-Gigleux, S, Magdalou, J, Burchell, B, Elbein, A D, Siest, G
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 15.07.1993
• Subjects: Adolescent; Adult; Affinity Labels; Animals; Bile Acids and Salts - metabolism; Catalysis; Cells, Cultured; Cricetinae; Deoxycholic Acid - metabolism; Female; Glucosidases - metabolism; Glucosides - analysis; Glucosides - biosynthesis; Glucosyltransferases - metabolism; Glucuronidase - metabolism; Humans; Kinetics; Male; Microsomes, Liver - metabolism; Middle Aged
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)82446-2

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.