Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases.
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.
Drake
R R
RR
Department of Biochemistry, University of Arkansas for Medical Sciences, Little Rock 72205.
Igari
Y
Y
Lester
R
R
Elbein
A D
AD
Radominska
A
A
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D010777:Photochemistry; D051381:Rats; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D003902:Detergents; 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; D005964:Glucosyltransferases; D014453:Glucuronosyltransferase; D010450:Endopeptidases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D014532:Uridine Diphosphate Glucose