Lee R, Monsey D, Weston A, Duncan K, Rithner C, McNeil M
Department of Microbiology, Colorado State University, Fort Collins 80523-1677, USA.
Anal Biochem. 1996 Nov 1;242(1):1-7. doi: 10.1006/abio.1996.0419.
A method to prepare UDP-galactofuranose (UDP-Galf) free of UDP-galactopyranose (UDP-Galp) is described. The UDP-Galf is synthesized enzymatically from UDP-Galp using the enzyme UDP-galactopyranose mutase. Treatment 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 its 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 spectrometry, and by several nuclear magnetic resonance techniques. In addition, an HPLC assay for the enzyme UDP-galactopyranose mutase is presented that requires 0.5 microgram 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 enzymes involved in galactofuranosyl biosynthesis for the cell walls of Mycobacteria, for the lipophosphoglycan of Leishmania, and for other microorganisms where galactofuranosyl residues are found.
描述了一种制备不含吡喃半乳糖基尿苷二磷酸（UDP-Galp）的呋喃半乳糖基尿苷二磷酸（UDP-Galf）的方法。UDP-Galf是使用UDP-吡喃半乳糖变位酶从UDP-Galp酶促合成的。用该酶处理UDP-Galp会产生UDP-Galp和UDP-Galf的平衡混合物，其中UDP-Galf约为7%。尽管产率较低，但使用戴安公司的PA-100离子交换高效液相色谱柱可轻松从起始的UDP-Galp中纯化出UDP-Galf。纯化后的UDP-Galf通过化学降解、电喷雾质谱法和几种核磁共振技术进行了表征。此外，还介绍了一种针对UDP-吡喃半乳糖变位酶的高效液相色谱分析方法，每次分析需要0.5微克UDP-Galf，可用于该酶活性的定性和定量测量。因此，这些方法应有助于对参与分枝杆菌细胞壁、利什曼原虫脂磷壁酸以及其他含有呋喃半乳糖基残基的微生物中呋喃半乳糖基生物合成相关酶的表征。
