Isotopolog perturbation techniques for metabolic networks:: Metabolic recycling of nutritional glucose in Drosophila melanogaster
Isotopolog perturbation techniques for metabolic networks:: Metabolic recycling of nutritional glucose in Drosophila melanogaster
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DOI：
10.1073/pnas.0400916101
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发表时间：
2004-04-27
影响因子：
11.1
通讯作者：
Bacher, A
中科院分区：
文献类型：
--
作者：
Eisenreich, W;Ettenhuber, C;Bacher, A
在添加了[U-C-13(6)]葡萄糖的标准培养基上培养黑胃果蝇（Drosophila melanogaster）菌株Oregon-R*。孵化后一到两天，用水将苍蝇提取出来。用层析法从提取物中分离出葡萄糖，并用C-13 NMR进行分析。所有的C-13信号都是由(CC)-C-13-C-13偶联产生的多重信号。基于对葡萄糖中耦合常数和重同位素位移的综合分析，单个C-13信号模式的积分提供了某些组C-13同位素的浓度。通过遗传算法对这些数据进行反卷积，得到所有单标记同位素和15个多标记同位素的丰度。在后一组中，有7种同位素在浓度为>0.1 mol %的情况下被发现，其中[1,2- c -13(2)]葡萄糖是最突出的同位素。多重c -13标记的葡萄糖同位素是由提供的葡萄糖通过复杂的分解代谢和合成代谢过程网络的代谢重塑引起的，这些过程包括糖酵解和/或通过戊糖磷酸、Cori循环和/或柠檬酸循环。所描述的微扰方法可以适用于各种各样的实验系统和同位素标记的前体。
Drosophila melanogaster strain Oregon-R* was grown on standard medium supplemented with [U-C-13(6)]glucose. One to two days after hatching, flies were extracted with water. Glucose was isolated chromatographically from the extract and was analyzed by C-13 NMR spectroscopy. All C-13 signals of the isolated glucose were multiplets arising by (CC)-C-13-C-13 coupling. Based on a comprehensive analysis of the coupling constants and heavy isotope shifts in glucose, the integrals of individual C-13 signal patterns afforded the concentrations of certain groups of C-13 isotopologs. These data were deconvoluted by a genetic algorithm affording the abundances of all single-labeled and of 15 multiply labeled isotopologs. Among the latter group, seven isotopologs were found at concentrations >0.1 mol % with [1,2-C-13(2)]glucose as the most prominent species. The Multiply C-13-labeled glucose isotopologs are caused by metabolic remodeling of the proffered glucose via a complex network of catabolic and anabolic processes involving glycolysis and/or passage through the pentose phosphate, the Cori cycle and/or the citrate cycle. The perturbation method described can be adapted to a wide variety of experimental systems and isotope-labeled precursors.