The preparation of UDP-N-acetylgalactosamine from UDP-N-acetylglucosamine employing UDP-N-acetylglucosamine-4-epimerase

A rapid, simple, and inexpensive method has been developed for preparing UDP-N-acetylgalactosamine in amounts sufficient for several thousand assays of enzymes that employ this nucleotide sugar as substrate. The UDP-N-acetylglucosamine-4-epimerase in extracts of porcine submaxillary glands was used to convert UDP-N-acetylglucosamine to an equilibrium mixture of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine (molar ratio, 77:23). The two nucleotide sugars were separated from components in the extract by ion-exchange chromatography and then separated from one another by affinity chromatography on a column of Griffonia simplicifolia lectin I bound to agarose. The UDP-N-acetylgalactosamine was obtained in pure form by ion-exchange chromatography in an overall yield of 91% from the equilibrium mixture. The separation of the two nucleotide sugars by affinity chromatography also provides a rapid assay for the UDPGlcNAc-4-epimerase, which is more accurate and less time consuming than earlier published assays.