5-azido-udp-glucuronic-acid and 5-azidouridine-5--diphosphoglucose

The photoaffinity analogues 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid have proven to be valuable biochemical tools in the studies of nucleoside diphosphate sugar-utilizing enzymes, especially membrane-associated glycosyltransferases. A summary of the past and current uses of these analogues is presented, as well as photoaffinity data for the enzyme UDP-glucose: dolichylphosphate glucosyltransferase (Glc-P-Dol synthase). This enzyme has served as a model membrane-associated glycosyltransferase for demonstrating the uses of 5-azido-UDP-glucose. The advantages of using photoaffinity analogues for the purification and characterization of glycosyltransferases are presented, as well as an outline of the general procedures which can be used in conjunction with these analogues.

Topics: Affinity Labels; Animals; Azides; Binding Sites; Glycosyltransferases; Humans; Molecular Structure; Photochemistry; Uridine Diphosphate Glucose; Uridine Diphosphate Glucuronic Acid

The photoaffinity analogs [beta-32P]5-azido-UDP-glucuronic acid ([32P]5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose ([32P]5N3UDP-Glc) were used to characterize UDP-glycosyltransferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to 56-kdalton range, known to contain UDP-glucuronosyl transferases (UGTs), was concentration dependent, and photolabeled proteins were susceptible to trypsin digestion only in the presence of detergent. The latter was demonstrated by the appearance on Western blots of the trypsin-treated, detergent-disrupted microsomes of a protein band of slightly lower molecular mass than, and presumably derived from, the UGTs. However, a labeled cleavage product was found only in samples photolabeled with [32P]5N3UDP-GlcUA and not in those labeled with [32P]5N3UDP-Glc. In detergent-treated microsomes, all of the nucleotide sugars that were tested protected better against photoinsertion of [32P]5N3UDP-GlcUA than of [32P]5N3UDP-Glc, with UDP-glucose being the most effective, followed by UDP-GlcUA and UDP-galactose. The pattern of inhibition of a series of uridinyl analogs toward photolabeling by the two probes was quite different: one inhibitor that was ineffective in blocking photoincorporation of [32P]5N3UDP-GlcUA (L-DPASiU) was one of the most potent inhibitors of photolabeling with [32P]5N3UDP-Glc. A similar dichotomy was seen with several inhibitors in enzymatic assays measuring hyodeoxycholic acid 6-O glucuronidation and glucosidation activities; the most potent inhibitors of HDCA glucosidation were not as effective against glucuronidation. The results indicate a lumenal orientation for human microsomal UGTs and provide substantial evidence that two distinct enzyme systems are involved in 6-O glucuronidation and 6-O glucosidation of HDCA.

Topics: Affinity Labels; Azides; Glycosyltransferases; Humans; Hydrolysis; Microsomes, Liver; Phosphorus Radioisotopes; Photochemistry; Trypsin; Uridine Diphosphate Glucose; Uridine Diphosphate Glucuronic Acid

Topics: Affinity Labels; Animals; Azides; Binding Sites; Electrophoresis, Polyacrylamide Gel; Fabaceae; Glycosyltransferases; Indicators and Reagents; Isotope Labeling; Liver; Macromolecular Substances; Phosphorus Radioisotopes; Plants, Medicinal; Swine; Uridine Diphosphate Glucose; Uridine Diphosphate Glucuronic Acid

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.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Affinity Labels; Animals; Azides; Biological Transport; Glucuronosyltransferase; Microsomes, Liver; Rats; Uridine Diphosphate Glucose; Uridine Diphosphate Glucuronic Acid

A new approach to determining the active site orientation of microsomal glycosyltransferases is presented which utilizes the photoaffinity analogs [32P]5-Azido-UDP-glucose ([32P]5N3UDP-Glc) and [32P]5-Azido-UDP-glucuronic acid ([32P]5N3UDP-GlcA). It was previously shown that both photoprobes could be used to photolabel UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), as well as the family of UDP-glucuronosyltransferases in rat liver microsomes. The effects of detergents, proteases, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the photolabeling of these enzymes were examined in intact rat liver microsomes. Photolabeling of Glc-P-Dol synthase by either photoprobe was the same in intact or disrupted vesicles, was susceptible to trypsin digestion, and was inhibited by the nonpenetrating inhibitor DIDS. Photolabeling of the UDP-glucuronosyltransferases by [32P]5N3UDP-GlcA was stimulated 1.3-fold in disrupted vesicles as compared to intact vesicles, whereas photolabeling of these enzymes by [32P]5N3UDP-Glc showed a 14-fold increase when vesicles were disrupted. Photolabeled UDP-glucuronosyltransferases were only susceptible to trypsin digestion in disrupted vesicles, and this was further verified by Western blot analyses. The results indicate a cytoplasmic orientation for access of UDP-sugars to Glc-P-Dol synthase and a lumenal orientation of most UDP-glucuronosyltransferases.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Affinity Labels; Animals; Azides; Binding Sites; Blotting, Western; Detergents; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Glucosyltransferases; Glucuronosyltransferase; Microsomes, Liver; Photochemistry; Rats; Uridine Diphosphate Glucose; Uridine Diphosphate Glucuronic Acid