Determination of different amino sugar 2'-epimerase activities by coupling to N-acetylneuraminate synthesis.
A new procedure for quantitating the amount of N-acetyl-D-mannosamine (ManNAc) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in sialic acid metabolism has been developed. The ManNAc generated by the action of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is condensed with pyruvate through the action of N-acetylneuraminate lyase and the sialic acid released is measured by the thiobarbituric acid assay. For the analysis of prokaryotic GlcNAc-6-phosphate 2'-epimerase, ManNAc-6-phosphate can also be evaluated by this coupled assay after dephosphorylation of the sugar phosphate. This system provides a sensitive, rapid, reproducible, specific and simple procedure (feasible with commercial reagents) for measuring amino sugar 2'-epimerases from eukaryotic and prokaryotic sources. The technique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and GlcNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epimerase in bacterial extracts.
Rodríguez-Aparicio
L B
LB
Departamento de Bioquímica y Biología Molecular, Universidad de León, Campus de Vegazana, 24007, León, Spain.
Ferrero
M A
MA
Revilla-Nuin
B
B
Martínez-Blanco
H
H
Reglero
A
A
D000606:Amino Sugars / Q000378:metabolism*; D000818:Animals; D001419:Bacteria; D002238:Carbohydrate Epimerases / Q000032:analysis* / Q000737:chemistry; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D007652:Oxo-Acid-Lyases / Q000737:chemistry; D051381:Rats; D012588:Scintillation Counting; D013053:Spectrophotometry / Q000379:methods; D013552:Swine; D013858:Thiobarbiturates
D000606:Amino Sugars; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D013858:Thiobarbiturates; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; C083982:wecB protein, E coli; C024519:N-acyl-D-glucosamine 2-epimerase; C498258:Renbp protein, rat; C121340:N-acylglucosamine-6-phosphate 2-epimerase; C029684:thiobarbituric acid