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 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 in time series experiments or inhibitor screens.
| Original language | English |
|---|---|
| Pages (from-to) | 94-100 |
| Number of pages | 7 |
| Journal | Analytical Biochemistry |
| Volume | 381 |
| Issue number | 1 |
| DOIs | |
| State | Published - Oct 1 2008 |
Funding
This work was supported in part by Public Health Service grant AI064444 from the National Institute of Allergy and Infectious Diseases and by the Petroleum Research Foundation (44382-G4). We thank Mehdi Moini and Lara Mahal for helpful discussions and also thank Susan Anderson for the gift of HeLa cells.
Keywords
- Endpoint assay
- Fluorescence
- Oxidoreductase
- Posttranslational glycosylation
- Sugar nucleotide
- UDP-N-acetyl-d-glucosamine