Abstract
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.
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