Monitoring of the proton electrochemical gradient in reconstituted vesicles: quantitative measurements of both transmembrane potential and intravesicular pH by ratiometric fluorescent probes
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Arylsulfonates chemistry MeSH
- Models, Biological * MeSH
- Cell Membrane chemistry physiology MeSH
- Electrochemistry MeSH
- Fluorescent Dyes analysis MeSH
- Spectrometry, Fluorescence MeSH
- Isoxazoles chemistry MeSH
- Hydrogen-Ion Concentration MeSH
- Membrane Potentials MeSH
- Proteolipids chemistry MeSH
- Proton-Translocating ATPases chemistry MeSH
- Protons * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Arylsulfonates MeSH
- Fluorescent Dyes MeSH
- Isoxazoles MeSH
- oxonol V MeSH Browser
- Proteolipids MeSH
- proteoliposomes MeSH Browser
- Proton-Translocating ATPases MeSH
- Protons * MeSH
- pyranine MeSH Browser
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 (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase. Its Deltapsi 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, Deltapsi and DeltapH. 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.
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