title: Characterization of UDP-glucuronic acid transport in rat liver microsomal vesicles with photoaffinity analogs.
abstract: The endoplasmic reticulum (ER) of rat liver contains several well characterized UDP-glucuronosyltransferases (UGTs), membrane-bound proteins of 50-54 kDa, and also less well identified UDP-glucosyltransferases, with nucleotide binding sites located on the lumenal surface. There is evidence that the substrates for these enzymes, UDP-glucuronic acid (UDP-GlcUA) and UDP-glucose (UDP-Glc), biosynthesized in the cytosol, are transported into the lumen of the ER via unknown mechanisms, the characteristics of which are poorly defined. A new approach for the study of the transport process has been devised using two active-site directed photoaffinity analogs, [beta-32P]5-azido-UDP-GlcUA and [beta-32P]5-azido-UDP-Glc. Photoincorporation of these probes into the lumenally oriented UGTs of intact rat liver microsomal vesicles was used as an indicator of transport. In intact vesicles, [32P]5N3UDP-GlcUA was efficiently incorporated into UGTs in a time, temperature and concentration dependent manner. In contrast, [32P]5N3UDP-Glc apparently was not transported effectively; maximal photolabeling of the 50-54 kDa proteins by this probe was dependent on detergent disruption of the vesicles. Vesicular uptake of and subsequent photolabeling of the 50-54 kDa proteins by [32P]5N3UDP-GlcUA were inhibited by UDP-GlcUA and 5N3UDP-GlcUA while UDP-Glc, 5N3UDP-Glc, UDP-xylose and UDP-N-acetylglucosamine were less inhibitory, suggesting a high degree of specificity for the uptake/photolabeling process. The anionic transport inhibitors DIDS and SITS inhibited [32P]5N3UDP-GlcUA photoincorporation into UGTs in intact vesicles, but also inhibited photolabeling of these and other enzymes in detergent disrupted vesicles. These data suggest the presence in rat liver microsomal vesicles of a specific, carrier-mediated transport process for UDP-GlcUA which is distinct from the mechanism of UDP-Glc transport.
authors.affiliation: Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
authors.forename: A
authors.initials: A
authors.lastname: Radominska
authors.forename: C
authors.initials: C
authors.lastname: Berg
authors.forename: S
authors.initials: S
authors.lastname: Treat
authors.forename: J M
authors.initials: JM
authors.lastname: Little
authors.forename: R
authors.initials: R
authors.lastname: Lester
authors.forename: J L
authors.initials: JL
authors.lastname: Gollan
authors.forename: R R
authors.initials: RR
authors.lastname: Drake
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001692:Biological Transport / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001386:Azides; C060227:5-azidouridine 5'-diphosphoglucose; C071932:5-azido-UDP-glucuronic acid; D014535:Uridine Diphosphate Glucuronic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D014453:Glucuronosyltransferase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D014532:Uridine Diphosphate Glucose