Quantitative analysis of H2O2 transport through purified membrane proteins
Quantitative analysis of H2O2 transport through purified membrane proteins
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H2O2 通过纯化膜蛋白转运的定量分析
DOI：
10.1016/j.mex.2020.100816
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发表时间：
2020-02
期刊：
影响因子：
1.9
通讯作者：
Kristina Hedfalk
中科院分区：
文献类型：
--
作者：
Hao Wang;Stefan Schoebel;Florian Schmitz;Hansong Dong;Kristina Hedfalk
过氧化氢（H2O2）是动物和植物细胞中产生的重要信号分子。细胞内和细胞外空间之间的 H2O2 平衡由整合膜蛋白调节，从而调节信号传导。已经建立了几种方法来分析全细胞中水通道蛋白介导的 H2O2 转运，其固有的局限性是负责某种活性的蛋白质的量无法标准化。因此，运输和特定活性的量化很难提取，这使得比较一个特定靶标的亚型和突变变体变得有问题。此外，在基于细胞的测定中，靶蛋白的表达可能会改变宿主细胞的生理过程，从而导致并发症和误导结果的风险。为了改进基于蛋白质的 H2O2 转运的测量，我们开发了一种允许定量测量的测定方法。•使用在蛋白脂质体中重构的纯化水通道蛋白，可以准确测量 H2O2 的转运。•在脂质体内，H2O2 催化 Amplex Red 和辣根过氧化物酶 (HRP) 之间的反应，产生荧光产物试卤灵。•分析纯蛋白质提供特定转运的直接生化证据排除假定的细胞背景。
Hydrogen peroxide (H2O2) is an important signal molecule produced in animal and plant cells. The balance of H2O2between the intra- and extracellular space is regulated by integral membrane proteins, which thereby modulate signaling. Several methods have been established to analyze aquaporin mediated transport of H2O2in whole cells with the intrinsic limitation that the amount of protein responsible for a certain activity cannot be standardized. As a consequence, the quantification of the transport and specific activity is difficult to extract making it problematic to compare isoforms and mutated variants of one specific target. Moreover, in cell-based assays, the expression of the target protein may alter the physiological processes of the host cell providing a complication and the risk of misleading results. To improve the measurements of protein based H2O2transport, we have developed an assay allowing quantitative measurements.•Using purified aquaporin reconstituted in proteoliposomes, transport of H2O2can be accurately measured.•Inside the liposomes, H2O2catalyzes the reaction between Amplex Red and horseradish peroxidase (HRP) giving rise to the fluorescent product resorufin.•Analysing pure protein provides direct biochemical evidence of a specific transport excluding putative cellular background.
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影响因子：
4.8
作者：
Bienert, Gerd P.;Moller, Anders L. B.;Jahn, Thomas P.
通讯作者：
Jahn, Thomas P.
影响因子：
15
作者：
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通讯作者：
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DOI：
10.1073/pnas.1116670109
发表时间：
2012-05-29
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
4.9
作者：
Hedfalk K
通讯作者：
Hedfalk K
影响因子：
48
作者：
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通讯作者：
Lukyanov, S