D-ARABINOSE SYNTHESIS AS A TARGET SITE FOR CHEMOTHERAPY

描述(摘自应用摘要)：D-阿拉伯糖是一种主要的 分枝杆菌细胞壁中的糖，存在于两个重要的细胞壁中 聚合物，阿拉伯半乳聚糖和脂阿拉伯甘露聚糖。尽管有 这种糖在这类生物体中广泛存在，很少 已知其生物合成途径。我们之前的研究 实验室和McNeil等人的实验室，其中分枝杆菌细胞 与[1-(14)C]或-[6-(14)C]葡萄糖孵育表明，两种标记 都是放射性阿拉伯糖的良好前体，这表明这 糖通过己糖前体的降解和重组而产生， 而不是直接通过己糖-一磷酸分流。然而， 具体步骤导致阿拉伯糖磷酸或活化形式的 D-阿拉伯糖是未知的，或者没有很好的文献记载。因此，虽然布伦南 已明确确定十一碳烯基-P-阿拉伯糖为 阿拉伯糖聚合物形成前的直接步骤 脂类还不清楚。已发表的关于核苷性质的报告 二磷酸阿拉伯糖是相互矛盾的。由于D-阿拉伯糖不存在于 任何动物细胞，其合成或激活的任何步骤都代表 极好的化疗靶点。因此，在这个试点项目中，我们 将探索参与合成和激活的反应 D-阿拉伯糖。首先，我们将分离和鉴定核苷 从分枝杆菌全细胞中提取二磷酸D-阿拉伯糖 确定核苷酸碱基的性质(文献中的一个报告 表示它是鸟苷，另一个表示它是尿苷)。在这些牢房里 提取物，我们还将寻找阿拉伯糖-5-P和 以验证阿拉伯糖-1-P的合成途径。然后，我们将孵化 分枝杆菌含放射性阿拉伯糖-5-P的无细胞提取物和 用阿拉伯糖-1-P检测是否存在磷酸阿拉伯糖变位酶和 XDP-阿拉伯糖焦磷酸化酶。这些酶将被提纯并 用各种潜在的抑制剂进行表征和测试，这些可能是 有用的化疗药物。

DESCRIPTION (adapted from application abstract): D-Arabinose is a major sugar of the mycobacterial cell wall and occurs in two important cell wall polymers, an arabinogalactan and a lipoarabinomannan. In spite of the widespread occurrence of this sugar in this group of organisms, very little is known about its pathway of biosynthesis. Previous studies from our laboratory and that of McNeil et al, in which mycobacterial cells were incubated with [1-(14)C]- or - [6-(14)C] glucose indicated that both labels were equally good precursors of radioactive arabinose, suggesting that this sugar arises via the degradation and reassembly of a hexose precursor, rather than directly by the hexose-monophosphate shunt. However, the specific steps leading to an arabinose-phosphate or an activated form of D-arabinose are unknown, or not well documented. Thus, while the Brennan group have definitely identified an undecaprenyl-P-arabinose as the immediate precuror of arabinose polymers, steps prior to formation of the lipid are unclear. Published reports on the nature of the nucleoside diphosphate arabinose are conflicting. Since D-arabinose does not occur in any animal cells, any of the steps in its synthesis or activation represent excellent target sites for chemotherapy. Thus, in this pilot project, we will explore the reactions involved in synthesis and activation of D-arabinose. First, we will isolate and characterize the nucleoside diphosphate D-arabinose from whole cells of mycobacteria, in order to determine the nature of the nucleotide base (one report in the literature indicates it is guanosine, another that it is uridine). In these cell extracts, we will also look for the presence of arabinose-5-P and arabinose-1-P to verify the pathway of synthesis. Then, we will incubate cell free extracts of mycobacteria with radioactive arabinose-5-P and arabinose-1-P to assay for the presence of a phosphoarabinose mutase and an XDP-arabinose pyrophosphorylase. These enzymes will be purified and characterized and tested with various potential inhibitors that may be useful chemotherapeutic agents.