A facile enzymatic synthesis of uridine diphospho-[14C]galacturonic acid.
A facile enzymatic synthesis of uridine diphospho-[14C]galacturonic acid.
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DOI：
10.1006/abio.2000.4479
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发表时间：
2000-04
影响因子：
2.9
通讯作者：
S. Basu;G. Dotson;C. Raetz
中科院分区：
文献类型：
--
作者：
S. Basu;G. Dotson;C. Raetz
半乳糖醛酸（GalA）是植物细胞壁来源的果胶的主要成分。它也可以在不同微生物的细胞表面多糖中发现，包括几种共生和致病细菌。尿苷二磷酸半乳糖醛酸（UDP-GalA）是这些多糖生物合成过程中GalA的可能供体。提出了一种高效、简便的UDP-[14 C]GalA的制备和纯化方法。在高水平半乳糖氧化酶和过氧化氢酶存在下，在延长的孵育时间下，将市售UDP-[14 C]-半乳糖定量氧化（>95%转化率）为UDP-[14 C]GalA。在该一步酶促氧化后，使用聚乙烯亚胺纤维素柱用单步1 M NaCl洗脱纯化UDP-[14 C]GalA。纯化的UDP-[14 C]GalA的真实性通过其在薄层色谱图上的相对迁移率、其化学水解产物的分析和1H NMR光谱来验证。我们的产率>90%，远高于先前描述的方法。该方法可作为制备其他放射性标记的糖醛酸及其核苷酸衍生物的原型。
Galacturonic acid (GalA) is a major component of plant cell-wall-derived pectins. It can be also found in the cell-surface polysaccharides of different microorganisms, including several symbiotic and pathogenic bacteria. Uridine diphosphogalacturonic acid (UDP-GalA) is a likely donor for GalA during the biosynthesis of these polysaccharides. A highly efficient, yet simple, method is presented for generating and purifying UDP-[14C]GalA. Commercially available UDP-[14C]-galactose was quantitatively oxidized (>95% conversion) to UDP-[14C]GalA in the presence of high levels of galactose oxidase and catalase, at prolonged incubation times. Following this one-step enzymatic oxidation, UDP-[14C]GalA was purified using a polyethyleneimine cellulose column with a single-step 1 M NaCl elution. The authenticity of the purified UDP-[14C]GalA was verified by its relative mobility on thin-layer chromatograms, analysis of its chemical hydrolysis products, and 1H NMR spectroscopy. Our yield of >90% is much higher than by previously described methods. The method may serve as a prototype for the preparation of other radiolabeled uronic acids and their nucleotide derivatives.