Radiometric assays of N-acetylglucosaminylphosphotransferase and α-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety
✍ Scribed by Yoav Ben-Yoseph; Michael S. Baylerian; Henry L. Nadler
- Book ID
- 102987145
- Publisher
- Elsevier Science
- Year
- 1984
- Tongue
- English
- Weight
- 684 KB
- Volume
- 142
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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.