ONE-DAY ENZYMATIC-SYNTHESIS AND PURIFICATION OF UDP-N-[1-C-14]ACETYL-GLUCOSAMINE

UDP-GlcN[1-C-14]Ac was synthesized in a single enzymatic reaction from [1-C-14]acetate and commercially available precursors on both a micro curie (micromole) and a millicurie (millimole) scale. The reaction was catalyzed by the action of acetyl coenzyme A synthetase, inorganic py rophosphatase, and the bifunctional Escherichia coli GlmU protein. Wit hin 2 h 86 to 94% reaction is attained, and it approaches 99% completi on overnight, GlmU protein was prepared in the form of a fusion suitab le for nickel chelate affinity chromatography. Several methods were de veloped for rapid purification of UDP-GlcN[1-C-14]Ac: an HPLC method h andled micromole (microcurie) loads, Alternatively, ion exchange chrom atography over DOWEX AG1 X-2 using a batch elution procedure was compa tible with millimole (millicurie) amounts of radiolabel and yielded bo th chemically and radiochemically homogeneous UDP-GlcN[1-C-14]Ac. Thes e methods allow laboratories to quickly produce and purify microcurie to millicurie quantities of N-acetyl-labeled UDP-GlcNAc by a choice of methods from relatively inexpensive precursors. (C) 1998 Academic Pre ss.