A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays:: Comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness
A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays:: Comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness
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DOI：
10.1105/tpc.104.025973
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发表时间：
2004-12-01
期刊：
影响因子：
11.6
通讯作者：
Stitt, M
中科院分区：
文献类型：
--
作者：
Gibon, Y;Blaesing, OE;Stitt, M
已经开发了一个平台来测量参与拟南芥中心碳和氮代谢的23种酶的活性。在优化的终止试验中测定活性，然后使用一套酶循环试验测定产物。该平台需要廉价的设备，以模块化方式组织以优化物流，自动计算结果，将高灵敏度与吞吐量相结合，可以自动化，并且具有每人/天100个样品中三到四个活动的吞吐量。几个测定，包括那些蔗糖磷酸合酶，ADP葡萄糖焦磷酸化酶（AGH 4），铁氧还蛋白依赖性谷氨酸合酶，甘油激酶，和莽草酸脱氢酶，提供了很大的优势，比以前的方法。该平台用于分析野生型哥伦比亚-0（Col-0）和无淀粉质体磷酸葡萄糖变位酶（pgm）突变体中的酶活性的昼夜变化，以及Col-0在夜间延长期间的酶活性的昼夜变化。将酶活性的变化与用Affyphase ATH 1阵列测定的转录水平的变化进行比较。转录水平的变化通常导致酶活性的强烈衰减变化。转录本的日变化幅度与酶活性之间没有相关性。AGEs和硝酸还原酶的活性日变化最大。检查的数据和与文献的比较表明，这些主要是因为翻译后调节。酶活性的变化也被强烈延迟，延迟因酶而异。有人提出，酶活性提供了一个准稳定的整合的监管在几个层面上，并提供有用的数据，为表征和诊断不同的生理状态。作为说明，使用来自Col-0的数据在昼夜变化和延长的黑暗处理期间构建的决策树用于显示，无论在昼夜循环期间的收获时间如何，pgm突变体类似于已经暴露于3d延长的夜晚的野生型植物。
A platform has been developed to measure the activity of 23 enzymes that are involved in central carbon and nitrogen metabolism in Arabidopsis thaliana. Activities are assayed in optimized stopped assays and the product then determined using a suite of enzyme cycling assays. The platform requires inexpensive equipment, is organized in a modular manner to optimize logistics, calculates results automatically, combines high sensitivity with throughput, can be robotized, and has a throughput of three to four activities in 100 samples per person/day. Several of the assays, including those for sucrose phosphate synthase, ADP glucose pyrophosphorylase (AGPase), ferredoxin-dependent glutamate synthase, glycerokinase, and shikimate dehydrogenase, provide large advantages over previous approaches. This platform was used to analyze the diurnal changes of enzyme activities in wild-type Columbia-0 (Col-0) and the starchless plastid phosphoglucomutase (pgm) mutant, and in Col-0 during a prolongation of the night. The changes of enzyme activities were compared with the changes of transcript levels determined with the Affymetrix ATH1 array. Changes of transcript levels typically led to strongly damped changes of enzyme activity. There was no relation between the amplitudes of the diurnal changes of transcript and enzyme activity. The largest diurnal changes in activity were found for AGPase and nitrate reductase. Examination of the data and comparison with the literature indicated that these are mainly because of posttranslational regulation. The changes of enzyme activity are also strongly delayed, with the delay varying from enzyme to enzyme. It is proposed that enzyme activities provide a quasi-stable integration of regulation at several levels and provide useful data for the characterization and diagnosis of different physiological states. As an illustration, a decision tree constructed using data from Col-0 during diurnal changes and a prolonged dark treatment was used to show that, irrespective of the time of harvest during the diurnal cycle, the pgm mutant resembles a wild-type plant that has been exposed to a 3 d prolongation of the night.