Measuring H+ pumping and membrane potential formation in sealed membrane vesicle systems
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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.
|Title of host publication||P-Type ATPases : methods and protocols|
|Number of pages||10|
|Publication status||Published - 2016|
|Series||Methods in Molecular Biology|
- 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