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 (ManN Ac) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in s ialic acid metabolism has been developed. The ManNAc generated by the actio n of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is conden sed with pyruvate through the action of N-acetylneuraminate lyase and the s ialic acid released is measured by the thiobarbituric acid assay. For the a nalysis 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, spe cific and simple procedure (feasible with commercial reagents) for measurin g amino sugar 2'-epimerases from eukaryotic and prokaryotic sources. The te chnique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and Gl cNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epim erase in bacterial extracts. (C) 1999 Elsevier Science B.V. All rights rese rved.