Monitoring of the proton electrochemical gradient in reconstituted vesicles: Quantitative measurements of both transmembrane potential and intravesicular pH by ratiometric fluorescent probes
Monitoring of the proton electrochemical gradient in reconstituted vesicles: Quantitative measurements of both transmembrane potential and intravesicular pH by ratiometric fluorescent probes
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DOI：
10.1007/s10895-007-0159-3
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发表时间：
2007-03-01
影响因子：
2.7
通讯作者：
Sigler, Karel
中科院分区：
文献类型：
--
作者：
Holoubek, Ales;Vecer, Jaroslav;Sigler, Karel
在其脂膜或质膜囊泡中携带重组酵母质膜 H+-ATP 酶的蛋白脂质体是方便研究涉及穿过微生物或植物细胞膜的质子电化学梯度 (Delta mu(+)(H)) 形成的基本电化学过程的模型系统。使用 Delta psi 和 pH 敏感荧光探针来监测重构囊泡的内部和外部体积之间形成的梯度。 Delta psi 敏感荧光比例探针 oxonol VI 适用于定量测量由重构的 H+-ATP 酶产生的内部阳性 Delta psi。其 Delta psi 响应可通过 K+/缬氨霉素方法进行校准，荧光测量的比率模式可减少不良的人为现象。原位 pH 敏感荧光探针吡喃用于定量测量蛋白脂质体内的 pH 值。对包埋在蛋白脂质体内的吡喃的 pH 敏感荧光响应进行校准，使用组合的几种离子载体来消除跨膜被动形成的梯度。所提出的模型系统提供了一个合适的工具，用于同时监测质子电化学梯度的两个组成部分：Delta psi 和 Delta pH。这种方法应该有助于进一步了解它们的形成如何在生物膜上相互关联，甚至其他离子的运输如何与其结合。
Proteoliposomes carrying reconstituted yeast plasma membrane H+-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Delta mu(+)(H)) across the microbial or plant cell membrane. Delta psi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Delta psi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Delta psi generated by the reconstituted H+-ATPase. Its Delta psi response can be calibrated by the K+/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Delta psi and Delta pH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.