phosphorus-radioisotopes and 5-azidouridine-5--diphosphoglucose

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

Photoaffinity labeling of purified cellulose synthase with [beta-32P]5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of [beta-32P]5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.

Topics: Affinity Labels; Arabidopsis Proteins; Autoradiography; Azides; Binding Sites; Cell Membrane; Gluconacetobacter xylinus; Glucosyltransferases; Macromolecular Substances; Molecular Weight; Phosphorus Radioisotopes; Uridine Diphosphate Glucose; Uridine Diphosphate Sugars

The radioactive, photoactivatable labeling probe [beta-32P]5-azidouridine 5'-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this radioactivity is due to labeling of phosphoglucomutase by contaminating levels of [32P]alpha Glc-1-P initially present at less than 1% of the total 32P. This conclusion is based in part on the ability of excess unlabeled alpha Glc-1-P and Glc-6-P, but not UDP-Glc, to block the labeling. In addition, the labeled protein in liver homogenates had a tryptic peptide pattern similar to that of authentic phosphoglucomutase. These findings, however, raised a second question. Assays for the UDP-Glc: glycoprotein glucosyl phosphotransferase (Glc phosphotransferase) have utilized [beta-32P]UDP-Glc and have resulted in the labeling of a small number of acceptors, including one of approximately 62 kDa. Despite the fact that these assays had routinely been performed in the presence of 1 mM alpha Glc-1-P, the coincidence in molecular weights led to these further studies. We conclude that the acceptor of approximately 62 kDa is distinct from phosphoglucomutase. This conclusion is based on differences in the time courses of incorporation, the specificity of blocking agents, the presence of covalently linked glucose, the products of acid hydrolysis and of beta-elimination, and isoelectric points.

Topics: Affinity Labels; Amino Acid Sequence; Animals; Azides; Binding Sites; Kinetics; Liver; Male; Molecular Sequence Data; Molecular Weight; Peptide Fragments; Phosphoglucomutase; Phosphorus Radioisotopes; Phosphotransferases; Proteins; Radioisotope Dilution Technique; Rats; Transferases (Other Substituted Phosphate Groups); Trypsin; Uridine Diphosphate Glucose; Uridine Diphosphate Sugars