Gibon Yves, Blaesing Oliver E, Hannemann Jan, Carillo Petronia, Höhne Melanie, Hendriks Janneke H M, Palacios Natalia, Cross Joanna, Selbig Joachim, Stitt Mark
Max Planck Institute of Molecular Plant Physiology, Science Park Golm, 14476 Golm-Potsdam, Germany.
Plant Cell. 2004 Dec;16(12):3304-25. doi: 10.1105/tpc.104.025973. Epub 2004 Nov 17.
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.
已开发出一个平台，用于测量拟南芥中参与中心碳和氮代谢的23种酶的活性。酶活性在优化的终止测定中进行检测，然后使用一套酶循环测定法来确定产物。该平台所需设备成本低廉，以模块化方式组织以优化物流，能自动计算结果，兼具高灵敏度和通量，可实现自动化操作，每人每天可对100个样品进行三到四项活性检测。其中一些测定方法，包括蔗糖磷酸合酶、ADP葡萄糖焦磷酸化酶（AGPase）、铁氧还蛋白依赖性谷氨酸合酶、甘油激酶和莽草酸脱氢酶的测定方法，相比以前的方法具有很大优势。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）突变体中酶活性的昼夜变化，以及Col-0在长夜延长期间的酶活性变化。将酶活性的变化与使用Affymetrix ATH1芯片测定的转录水平变化进行比较。转录水平的变化通常导致酶活性变化的大幅衰减。转录本和酶活性的昼夜变化幅度之间没有关联。AGPase和硝酸还原酶的活性昼夜变化最大。对数据的检查以及与文献的比较表明，这些主要是由于翻译后调控。酶活性的变化也有很强的延迟，不同酶的延迟各不相同。有人提出，酶活性在多个水平上提供了一种准稳定的调控整合，并为不同生理状态的表征和诊断提供了有用的数据。作为一个例证，使用Col-0在昼夜变化和长时间黑暗处理期间的数据构建的决策树表明，无论在昼夜周期中的收获时间如何，pgm突变体类似于已接受3天长夜延长处理的野生型植物。
