Method for the determination of cellular levels of guanosine-5'-diphosphate-mannose based on a weak interaction with concanavalin A at low pH.
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.
Rush
J S
JS
Department of Biochemistry, A. B. Chandler Medical Center, University of Kentucky, Lexington 40536, USA.
Waechter
C J
CJ
D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells / Q000737:chemistry / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D006224:Cricetinae; D005260:Female; D005910:Glioma / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008213:Lymphocyte Activation / Q000502:physiology; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D011105:Polyisoprenyl Phosphate Sugars / Q000096:biosynthesis; D051381:Rats; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000031:analogs & derivatives*; D014316:Tritium
C066723:Glc(3)Man(9)(GlcNAc)(2)-diphosphate-dolichol; D006023:Glycoproteins; D009711:Nucleotides; D011105:Polyisoprenyl Phosphate Sugars; C028326:concanavalin A-sepharose; D014316:Tritium; D006155:Guanosine Diphosphate Mannose; D012685:Sepharose; D008358:Mannose