ENZYMATIC-SYNTHESIS OF UDP-GALACTOFURANOSE AND AN ASSAY FOR UDP-GALACTOPYRANOSE MUTASE BASED ON HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A method to prepare UDP-galactofuranose (UDP-Galf) free of UDP-galacto pyranose (UDP-Galp) is described, The UDP-Galf is synthesized enzymati cally from UDP-Galp using the enzyme UDP-galactopyranose mutase. Treat ment of UDP-Galp with the enzyme yields an equilibrium mixture of UDP- Galp and UDP-Galf in which UDP-Galf is approximately 7%. In spite of i ts low yield, the UDP-Galf is readily purified from starting UDP-Galp using a Dionex PA-100 ion exchange HPLC column, The purified UDP-Galf was characterized by chemical degradations, by electrospray mass spect rometry, and by several nuclear magnetic resonance techniques, In addi tion, an HPLC assay for the enzyme UDP-galactopyranose mutase is prese nted that requires 0.5 mu g of UDP-Galf per assay and can be used for both qualitative and quantitative measurements of the enzyme activity, These procedures should thus aid in the characterization of the enzym es involved in galactofuranosyl biosynthesis for the cell, walls of My cobacteria, for the lipophosphoglycan of Leishmania, and for other mic roorganisms where galactofuranosyl residues are found. (C) 1996 Academ ic Press, Inc.