Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system
Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system
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DOI：
10.1046/j.1365-313x.2001.01278.x
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发表时间：
2002-04-01
期刊：
影响因子：
7.2
通讯作者：
Stitt, M
中科院分区：
文献类型：
--
作者：
Gibon, Y;Vigeolas, H;Stitt, M
需要代谢物测定来确定发育过程中基因型之间的代谢变化以及对环境扰动的响应。它们提供了一个跳板，以确定重要的监管网站和调查的潜在机制。由于它们的小尺寸，拟南芥种子对这种测量提出了技术挑战。一套基于甘油-3-磷酸脱氢酶和甘油-3-磷酸氧化酶之间的新型酶循环系统的测定方法已经开发并优化用于生长拟南芥种子。结合现有的检测方法，它们为淀粉（腺嘌呤二磷酸葡萄糖）和脂质（乙酰辅酶A，甘油-3-磷酸）合成的直接前体以及焦磷酸盐，ATP，ADP和大多数糖酵解中间体提供了一套高通量，灵敏的检测方法。还提出了一种方法来快速淬灭完整的长角果，冻干它们，然后手动分离种子进行代谢物分析。这些技术被用来调查在整个种子代谢物水平的变化，在发展和成熟过程中，并在逐步减少外部氧浓度的反应。
Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.