CHARACTERIZATION OF HUMAN LIVER MICROSOMAL UDP-GLYCOSYLTRANSFERASES USING PHOTOAFFINITY ANALOGS

The photoaffinity analogs [beta-P-32]5-azido-UDP-glucuronic acid ([P-3 2]5N3UDP-GlcUA) and [beta-P-32]5-azido-UDP-glucose ([P-32]5N(3)UDP-Glc ) were used to characterize UDP-glycosyl-transferases of microsomes pr epared from human liver. Photoincorporation of both probes into protei ns in the 50- to 56-kdalton range, known to contain UDP-glucuronosyl t ransferases (UGTs), was concentration dependent, and photolabeled prot eins were susceptible to trypsin digestion only in the presence of det ergent. The latter was demonstrated by the appearance on Western blots of the trypsin-treated, detergent-disrupted microsomes of a protein b and of slightly lower molecular mass than, and presumably derived from , the UGTs. However, a labeled cleavage product was found only in samp les photolabeled with [P-32]5N(3)UDP-GlcUA and not in those labeled wi th [P-32]5N(3)UDP-Glc. In detergent-treated microsomes, all of the nuc leotide sugars that were tested protected better against photoinsertio n of [P-32]5N(3)UDP-GlcUA than of [P-32]5N(3)UDP-Glc, with UDP-glucose being the most effective, followed by UDP-GlcUA and UDP-galactose. Th e pattern of inhibition of a series of uridinyl analogs toward photola beling by the two probes was quite different: one inhibitor that was i neffective in blocking photoincorporation of [P-32]5N(3)UDP-GlcUA (L-D PASiU) was one of the most potent inhibitors of photolabeling with [P- 32]5N(3)UDP-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 gluco sidation were not as effective against glucuronidation. The results in dicate a lumenal orientation for human microsomal UGTs and provide sub stantial evidence that two distinct enzyme systems are involved in 6-O glucuronidation and 6-O glucosidation of HDCA.