CHARACTERIZATION OF UDP-GLUCURONIC ACID TRANSPORT IN RAT-LIVER MICROSOMAL VESICLES WITH PHOTOAFFINITY ANALOGS

The endoplasmic reticulum (ER) of rat liver contains several well char acterized 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 i s 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 c haracteristics of which are poorly defined. A new approach for the stu dy of the transport process has been devised using two active-site dir ected photoaffinity analogs, [beta-P-32]5-azido-UDP-GlcUA and [beta-P- 32]5-azido-UDP-Glc. Photoincorporation of these probes into the lumena lly oriented UGTs of intact rat liver microsomal vesicles was used as an indicator of transport. In intact vesicles, [P-32]5N(3)UDP-GlcUA wa s efficiently incorporated into UGTs in a time, temperature and concen tration dependent manner. In contrast, [P-32]5N(3)UDP-Glc apparently w as not transported effectively; maximal photolabeling of the 50-54 kDa proteins by this probe was dependent on detergent disruption of the v esicles. Vesicular uptake of and subsequent photolabeling of the 50-54 kDa proteins by [P-32]5N(3)UDP-GlcUA were inhibited by UDP-GlcUA and 5N(3)UDP-GlcUA while UDP-Glc, 5N(3)UDP-Glc, UDP-xylose and UDP-N-acety lglucosamine were less inhibitory, suggesting a high degree of specifi city for the uptake/photolabeling process. The anionic transport inhib itors DIDS and SITS inhibited [P-32]5N(3)UDP-GlcUA photoincorporation into UGTs in intact vesicles, but also inhibited photolabeling of thes e and other enzymes in detergent disrupted vesicles. These data sugges t the presence in rat liver microsomal vesicles of a specific, carrier -mediated transport process for UDP-GlcUA which is distinct from the m echanism of UDP-Glc transport.