Radiometric assays of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety.
The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety.
D001665:Binding Sites; D002250:Carbon Radioisotopes; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D002854:Chromatography, Paper; D005347:Fibroblasts / Q000201:enzymology; D005944:Glucosamine; D006801:Humans; D007553:Isotope Labeling; D007962:Leukocytes / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000032:analysis*; D010770:Phosphotransferases / Q000032:analysis*; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups); D014316:Tritium
D002250:Carbon Radioisotopes; D014316:Tritium; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase; D005944:Glucosamine