A rapid and simple two-step procedure for the quantitative analysis of
GDP-mannose (
GDP-Man) recovered in
ethanol extracts of cultured mammalian cells is described.
GDP-Man is initially separated from water-soluble metabolites and other
nucleotide sugars, including
UDP-glucose (
UDP-Glc) and
GDP-fucose (
GDP-Fuc), due to a weak, alpha-
mannoside-specific interaction with
concanavalin A (
Con A)-Sepharose at pH 3.5. The specificity and pH dependence of the
GDP-Man-Con A interaction have been characterized. The partially purified fraction from
Con A-Sepharose can be further purified by high-performance
anion-exchange chromatography on a Partisil-10 SAX
silica gel column, and the concentration of
GDP-Man was determined by monitoring the HPLC column eluate for absorbance at 254 nm. This procedure provides a simple means of calculating the specific activity of cellular
GDP-[3H]Man pools, metabolically labeled with [2-3H]
mannose. Using this new procedure, the relative rates of Glc3Man9Glc-NAc2-P-P-dolichol (
Oligo-P-P-Dol) biosynthesis and
protein N-glycosylation were assayed in C 6 rat glial
tumor cells, COS P6 cells, Chinese hamster ovary (CHO) cells, and mouse L929 cells by metabolic labeling with [2-3H]
mannose. A comparison of the relative rates of incorporation of [2-3H]
mannose into
Oligo-P-P-Dol and N-linked
oligosaccharides in four different cultured cell lines demonstrates that misleading results can be obtained if the calculation of the biosynthetic rates is not based on the specific activity of the
nucleotide sugar pools.