A novel UDP-Glc-specific glucosyltransferase catalyzing the biosynthesis of 6-O-glucosides of bile acids in human liver microsomes.
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.
Radominska
A
A
Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
Little
J
J
Pyrek
J S
JS
Drake
R R
RR
Igari
Y
Y
Fournel-Gigleux
S
S
Magdalou
J
J
Burchell
B
B
Elbein
A D
AD
Siest
G
G
D000293:Adolescent; D000328:Adult; D000345:Affinity Labels; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002384:Catalysis; D002478:Cells, Cultured; D006224:Cricetinae; D003840:Deoxycholic Acid / Q000378:metabolism; D005260:Female; D005959:Glucosidases / Q000378:metabolism; D005960:Glucosides / Q000032:analysis / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008875:Middle Aged
D000345:Affinity Labels; D001647:Bile Acids and Salts; D005960:Glucosides; D003840:Deoxycholic Acid; C010471:hyodeoxycholic acid; D005964:Glucosyltransferases; D005959:Glucosidases; D005966:Glucuronidase