Pauly M, Porchia A, Olsen C E, Nunan K J, Scheller H V
Plant Biochemistry Laboratory, Department of Chemistry, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, Copenhagen, Denmark.
Anal Biochem. 2000 Feb 1;278(1):69-73. doi: 10.1006/abio.1999.4411.
Many plant cell wall components such as the polysaccharides xylans and pectins or the glycoproteins arabinogalactan proteins and extensins contain arabinosyl residues. The arabinosyl substituents are thought to be incorporated into these wall polymers by the action of arabinosyltransferases using UDP-l-arabinose as the precursor. UDP-l-arabinose is not commercially available and therefore a procedure for generating UDP-l-arabinose was developed for use in studies on the biosynthesis of the arabinose-containing polymers. In this procedure UDP-d-xylose is incubated with an enzyme preparation from wheat germ and the nucleotide sugars in the reaction mixture are extracted. High-performance anion-exchange chromatography of the extract resolves two major UV-absorbing components: one corresponding to UDP-xylose and a second that elutes earlier. TLC analysis of collected and hydrolyzed fractions demonstrated the presence of l-arabinose in the early eluting fraction. Further analysis by NMR identified the compound as UDP-beta-l-arabinopyranose. The procedure reported here provides an efficient method for preparing either radioactive UDP-l-[(14)C]arabinose or nonradioactive UDP-l-arabinose and can also be used as an assay for UDP-xylose-4-epimerase activity.
许多植物细胞壁成分，如木聚糖和果胶等多糖或阿拉伯半乳聚糖蛋白和伸展蛋白等糖蛋白，都含有阿拉伯糖基残基。阿拉伯糖基取代基被认为是通过阿拉伯糖基转移酶的作用，以UDP-L-阿拉伯糖为前体掺入这些细胞壁聚合物中的。UDP-L-阿拉伯糖没有商业供应，因此开发了一种生成UDP-L-阿拉伯糖的方法，用于含阿拉伯糖聚合物生物合成的研究。在该方法中，将UDP-D-木糖与来自小麦胚芽的酶制剂一起温育，并提取反应混合物中的核苷酸糖。提取物的高效阴离子交换色谱分离出两个主要的紫外线吸收成分：一个对应于UDP-木糖，另一个洗脱较早。对收集并水解的馏分进行TLC分析表明，较早洗脱的馏分中存在L-阿拉伯糖。通过NMR进一步分析确定该化合物为UDP-β-L-阿拉伯吡喃糖。本文报道的方法为制备放射性UDP-L-[(14)C]阿拉伯糖或非放射性UDP-L-阿拉伯糖提供了一种有效的方法，也可用于UDP-木糖-4-表异构酶活性的测定。
