Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine.
Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine.
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尿苷二磷酸 N-乙酰基-D-葡萄糖胺的酶法分析。
DOI：
10.1016/j.ab.2008.06.034
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发表时间：
2008
影响因子：
2.9
通讯作者：
Graham,DavidE
中科院分区：
文献类型：
--
作者：
Namboori,SeemaC;Graham,DavidE
Maripaludis MMP0352蛋白属于氧化还原酶家族，催化尿苷二磷酸N-乙酰-d-氨基葡萄糖(UDP-GlcNAc)3‘端NAD+依赖的氧化，形成3-己糖核苷酸。对异源表达的MMP0352蛋白进行了纯化，结果表明MMP0352蛋白能有效催化UDP-GlcNAc氧化，形成一个NADH等效物。利用该酶建立了一种分析UDP-GlcNAc的固定终点荧光法。该酶对该乙酰氨基糖核苷酸具有高度的特异性，在1毫升样品中的检出限为0.2UDP-μ-GlcNAc。采用标准加入法，测定了大肠杆菌、酿酒酵母和HeLa癌细胞去蛋白提取物中UDP-GlcNAc的浓度。通过酶分析和层析分析确定了当量浓度，验证了该方法的有效性。该方法可用于时间序列实验或抑制剂筛选中细胞UDP-GlcNAc浓度变化的高通量分析。
The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD+-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP–GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP–GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP–GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2μM UDP–GlcNAc in a 1-ml sample. Using the method of standard addition, UDP–GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP–GlcNAc concentrations in time series experiments or inhibitor screens.