Quantification of NAD(P)H in cyanobacterial cells by a phenol extraction method.
Quantification of NAD(P)H in cyanobacterial cells by a phenol extraction method.
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DOI：
10.1007/s11120-021-00835-1
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发表时间：
2021-05
影响因子：
3.7
通讯作者：
Nakanishi S
中科院分区：
文献类型：
--
作者：
Tanaka K;Shimakawa G;Tabata H;Kusama S;Miyake C;Nakanishi S
在光合生物中，人们认识到NADPH的细胞内氧化还原比被调节在适当的范围内，以实现多种生理过程的协同功能。然而，尽管它很重要，但迄今为止定量估计的 NADPH 分数 [NADPH/(NADPH + NADP+)] 值存在很大差异。在本研究中，通过应用一种新的 NADP(H) 提取方法，使用苯酚/氯仿/异戊醇 (PCI) 在蓝藻集胞藻中研究 NADPH 组分的光响应。 PCC 6803。使用 PCI 提取观察到的 NADP(H) 光响应与体内测量的 NAD(P)H 荧光时间过程定性一致。此外，通过 PCI 提取和基于荧光的方法获得的结果在呼吸链中缺乏氧化 NAD(P)H 的能力并表现出独特的 NADPH 光响应的突变体中也一致。 These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light–dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions. The online version contains supplementary material available at 10.1007/s11120-021-00835-1.
In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP+)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light–dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions. The online version contains supplementary material available at 10.1007/s11120-021-00835-1.
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DOI：
10.1007/978-1-4939-0473-0_26
发表时间：
2014-01-01
期刊：
PSEUDOMONAS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Kern, Suzanne E.;Price-Whelan, Alexa;Newman, Dianne K.
通讯作者：
Newman, Dianne K.
影响因子：
2.9
作者：
BADGER, MR;LORIMER, GH
通讯作者：
LORIMER, GH
影响因子：
4.3
作者：
Latouche, G;Cerovic, ZG;Moya, I
通讯作者：
Moya, I
影响因子：
6.9
作者：
Takahashi H;Uchimiya H;Hihara Y
通讯作者：
Hihara Y
影响因子：
4.8
作者：
Ohkawa, H;Pakrasi, HB;Ogawa, T
通讯作者：
Ogawa, T