Radiometric assays of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety |
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Authors: | Y Ben-Yoseph M S Baylerian H L Nadler |
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Institution: | Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128 USA |
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Abstract: | 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. |
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Keywords: | electrophoresis SDS Coomassie blue photography polyacrylamide slab gel |
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