Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water.
N-Acetyl-D-[2-3H]glucosamine was synthesized from N-acetyl-D-mannosamine by alkaline 2-epimerization in pyridine containing 3H2O and nickelous acetate. The reaction involves reversible formation of an enol intermediate and therefore also resulted in incorporation of tritium into N-acetylmannosamine. After completed reaction, the two N-acetylhexosamines were separated from other radioactive products and Morgan-Elson chromogens by chromatography on a column of Sephadex G-10, which was eluted with 10% ethanol, and were then separated from each other by chromatography on Sephadex G-15 in 0.27 M sodium borate (pH 7.8). The location of the incorporated tritium was established by treatment of the N-acetylhexosamines with borate under the conditions of the Morgan-Elson reaction, which converts the sugars to Kuhn's chromogen I with concomitant loss of the C-2 hydrogen. As expected, this treatment resulted in the formation of 3H2O, indicating that the tritium was located at C-2. [2-3H]Glucosamine was prepared by acid hydrolysis of the labelled N-acetylglucosamine and was converted to [2-3H]glucosamine 6-phosphate by incubation with hexokinase and ATP. The sugar phosphate was used as a substrate for glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10) in a simple 3H2O release assay.
Rodén
L
L
School of Medicine, University of Alabama at Birmingham 35294, USA.
Jin
J
J
Yu
H
H
Campbell
P
P
D000117:Acetylglucosamine / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D019747:Aldose-Ketose Isomerases; D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002854:Chromatography, Paper; D004590:Electrophoresis, Paper; D006595:Hexosamines; D006358:Hot Temperature; D006868:Hydrolysis; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014316:Tritium; D014867:Water
D006595:Hexosamines; D007202:Indicators and Reagents; D014867:Water; D014316:Tritium; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; D000117:Acetylglucosamine; C002022:N-acetylmannosamine