Ben-Yoseph Y, Baylerian M S, Nadler H L
Anal Biochem. 1984 Nov 1;142(2):297-304. doi: 10.1016/0003-2697(84)90468-8.
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.
利用α-甲基甘露糖苷受体和市售的UDP-[³H或¹⁴C]N-乙酰葡糖胺供体对成纤维细胞和白细胞N-乙酰葡糖胺磷酸转移酶进行测定，通过额外的洗脱步骤进行了改进，以产生低背景，从而获得与使用合成制备的[β-³²P]UDP-N-乙酰葡糖胺供体所获得的结果相当的高灵敏度和可靠性。这是通过一个额外的洗脱步骤实现的，该步骤去除了游离的[³H或¹⁴C]N-乙酰葡糖胺，后者似乎是导致高背景的分解产物。此外，转移反应的[³H或¹⁴C]N-乙酰葡糖胺-1-磷酸-6-α-甲基甘露糖苷产物随后被分离，脱盐后可作为α-N-乙酰葡糖胺磷酸二酯酶测定的底物。I-细胞病和假胡尔勒氏多营养不良患者的细胞制剂对葡糖胺部分标记有³H或¹⁴C的相应底物表现出严重至中度的转移酶活性缺乏和正常的磷酸二酯酶活性。
