Characterization of human liver microsomal UDP-glycosyltransferases using photoaffinity analogs.
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.
Little
J M
JM
Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, USA.
Drake
R R
RR
Vonk
R
R
Kuipers
F
F
Lester
R
R
Radominska
A
A
D000345:Affinity Labels; D001386:Azides; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008862:Microsomes, Liver / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D014357:Trypsin / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives
D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; C060227:5-azidouridine 5'-diphosphoglucose; C071932:5-azido-UDP-glucuronic acid; D014535:Uridine Diphosphate Glucuronic Acid; D016695:Glycosyltransferases; D014357:Trypsin; D014532:Uridine Diphosphate Glucose