Enzymatic analysis of UDP-N-acetylglucosamine

Enzymatic analysis of UDP-N-acetylglucosamine

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 MM...

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Veröffentlicht in:Analytical biochemistry 2008-06, Vol.381 (1), p.94-100
Hauptverfasser: Namboori, Seema C., Graham, David E.
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Graham, David E.
description 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 0.2 μM 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 during time series experiments or inhibitor screens.
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title Enzymatic analysis of UDP-N-acetylglucosamine
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