Abstract
The activity of enzymes involved in active transport of matter across lipid bilayers can conveniently be assayed by measuring their consumption of energy, such as ATP hydrolysis, while it is more challenging to directly measure their transport activities as the transported substrate is not converted into a product and only moves a few nanometers in space. Here, we describe two methods for the measurement of active proton pumping across lipid bilayers and the concomitant formation of a membrane potential, applying the dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI. The methods are exemplified by assaying transport of the Arabidopsis thaliana plasma membrane H+-ATPase (proton pump), which after heterologous expression in Saccharomyces cerevisiae and subsequent purification has been reconstituted in proteoliposomes.
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Acknowledgement
This work was supported by the UNIK research initiative of the Danish Ministry of Science, Technology and Innovation through the “Center for Synthetic Biology” at the University of Copenhagen and the Danish National Research Foundation through the PUMPKIN Center of Excellence (DNRF85).
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This work is dedicated to the memory of Alex Green Wielandt, who set the basis of this study until his untimely death.
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Wielandt, A.G., Palmgren, M.G., Fuglsang, A.T., Günther-Pomorski, T., Justesen, B.H. (2016). Measuring H+ Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_17
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DOI: https://doi.org/10.1007/978-1-4939-3179-8_17
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