Unknown

Dataset Information

0

Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine.


ABSTRACT: 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 microM 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.

SUBMITTER: Namboori SC 

PROVIDER: S-EPMC2597562 | biostudies-other | 2008 Oct

REPOSITORIES: biostudies-other

altmetric image

Publications

Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine.

Namboori Seema C SC   Graham David E DE  

Analytical biochemistry 20080627 1


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  ...[more]

Similar Datasets

| S-EPMC4498474 | biostudies-literature
| S-EPMC3272127 | biostudies-literature
| S-EPMC8089376 | biostudies-literature
| S-EPMC3050423 | biostudies-literature
| S-EPMC3391414 | biostudies-literature
| S-EPMC4427165 | biostudies-literature
| S-EPMC3219419 | biostudies-literature
| S-EPMC9299652 | biostudies-literature
| S-EPMC8470988 | biostudies-literature
| S-EPMC7558457 | biostudies-literature