Measuring H+ pumping and membrane potential formation in sealed membrane vesicle systems

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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.

Original languageEnglish
Title of host publicationP-Type ATPases : methods and protocols
EditorsMaike Bublitz
Number of pages10
VolumePart II
Publication date2016
ISBN (Print)978-1-4939-3178-1
ISBN (Electronic)978-1-4939-3179-8
Publication statusPublished - 2016
SeriesMethods in Molecular Biology

    Research areas

  • Adenosine Triphosphate, Aminoacridines, Arabidopsis, Biological Transport, Active, Gene Expression Regulation, Enzymologic, Hydrolysis, Isoxazoles, Lipid Bilayers, Membrane Potentials, Proton-Translocating ATPases, Saccharomyces cerevisiae, Journal Article, Research Support, Non-U.S. Gov't

ID: 169733840