叶绿体NAD激酶活性的测定及全组织NAD激酶分析
Measurement of Chloroplastic NAD Kinase Activity and Whole Tissue NAD Kinase Assay.
作者信息
Ishikawa Yuuma, Kawai-Yamada Maki, Hashida Shin-Nosuke
机构信息
Graduate School of Science and Engineering, Saitama University, Saitama, Japan.
Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, Abiko, Japan.
出版信息
Bio Protoc. 2020 Jan 5;10(1):e3480. doi: 10.21769/BioProtoc.3480.
Nicotinamide adenine dinucleotide phosphate (NADP) synthesis requires nicotinamide adenine dinucleotide (NAD) kinase activity, substrate NAD and ATP. The NAD kinase responds to various environmental stimuli and its activity is regulated via various regulatory pathways, such as Ca-dependent and redox-dependent signals. Conventional NAD kinase assay has been useful to evaluate enzyme activity; however, recent reports revealed a dynamics of NADP pool (the sum of NADP and NADPH) under fluctuating light condition, indicating that the rate of NADP synthesis is not always determined by NAD kinase activity. Here, we developed a novel method for the estimation of chloroplastic NAD kinase activity by quantifying the changes in the NADP amounts in response to illumination. As our approach does not involve protein extraction, it saves time (compared to the assay), thereby allowing for a sequence of assays, and provides several clues in the investigation of regulatory mechanisms behind NADP synthesis under various environmental conditions.
烟酰胺腺嘌呤二核苷酸磷酸（NADP）的合成需要烟酰胺腺嘌呤二核苷酸（NAD）激酶活性、底物NAD和ATP。NAD激酶对各种环境刺激作出反应，其活性通过多种调节途径进行调节，如钙依赖性和氧化还原依赖性信号。传统的NAD激酶测定法对于评估酶活性很有用；然而，最近的报告揭示了波动光照条件下NADP库（NADP和NADPH的总和）的动态变化，这表明NADP的合成速率并不总是由NAD激酶活性决定。在这里，我们开发了一种新方法，通过量化光照响应下NADP量的变化来估算叶绿体NAD激酶活性。由于我们的方法不涉及蛋白质提取，节省了时间（与测定法相比），从而可以进行一系列测定，并为研究各种环境条件下NADP合成背后的调节机制提供了几条线索。
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