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 commerically 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.