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Development of Chemical Analysis for Metabolic Intermediates

代谢中间体化学分析的进展

基本信息

批准号：
12206006
负责人：
NISHIOKA Takaaki
金额：
$ 57.09万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

项目摘要

Most of the metabolites in living cells or tissues are polar and ionic substances that are not suitable for chemical analysis by using conventional methods such as GC-and LC-MS. We have successfully developed capillary electrophoresis coupled with mass spectrometry (CE-MS) as a method of metabolomics. We applied the method to analyze the metabolic intermediates of the central energy metabolism of the Bacillus subtilis cells and compared the metabolite profiles that schematically show the intercellular amount of the metabolites on the central metabolic pathways of the cells cultured under various environmental and genetic perturbations. The metabolite profiles were globally same when the rates of the cell division were the same under the different perturbations. This suggests that B. subtilis has a metabolite profile that is optimized for its cell division and that it maintain the profile against the environmental and genetic perturbations. At the same times when we analyzed the metabolites, we measured the gene expressions by using the DNA microarray of B. subtilis. Gene expression data were overlaid on the metabolite profiles. When perturbations were not so different from the control experiment, amount of the expressions of several enzyme genes were regulated. On the others where perturbations were quite different, B. subtilis changed not only gene expressions but also metabolic pathways; it used a quite different metabolic pathways from those of the control experiment. These results suggests that the metabolite profile is quantitatively and qualitatively regulated; this must be a way of "homeostasis". It is most likely that each living species has its own metabolite profile for cell division.

活细胞或组织中的代谢物大多是极性和离子物质，不适合用GC-MS和LC-MS等常规方法进行化学分析。我们已经成功地建立了毛细管电泳质谱(CE-MS)作为代谢组学的一种方法。我们应用该方法分析了枯草芽孢杆菌细胞中心能量代谢的代谢中间产物，并比较了在各种环境和遗传扰动下培养的细胞的中心代谢途径上代谢物的胞间数量。当细胞分裂速率在不同扰动下相同时，代谢物分布总体上是相同的。这表明枯草杆菌的代谢物图谱是为其细胞分裂而优化的，并且它在环境和遗传扰动下保持了这种图谱。在分析代谢产物的同时，我们利用枯草杆菌DNA微阵列检测了基因的表达。基因表达数据覆盖在代谢物图谱上。在扰动与对照相差不大的情况下，几种酶基因的表达受到了调控。在其他扰动完全不同的地方，枯草芽孢杆菌不仅改变了基因表达，还改变了代谢途径；它使用了与对照实验完全不同的代谢途径。这些结果表明，代谢物的分布在数量和质量上都得到了调节；这肯定是一种“内稳态”的方式。最有可能的是，每个活着的物种都有自己的细胞分裂代谢物图谱。