title: Measuring H(+) Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems.
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.
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Alex Green
authors.initials: AG
authors.lastname: Wielandt
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Palmgren
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Anja Thoe
authors.initials: AT
authors.lastname: Fuglsang
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Günther-Pomorski
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark. bjust@plen.ku.dk.
authors.forename: Bo Højen
authors.initials: BH
authors.lastname: Justesen
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000737:chemistry*; D000609:Aminoacridines / Q000737:chemistry; D017360:Arabidopsis / Q000201:enzymology; D001693:Biological Transport, Active; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D007555:Isoxazoles / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008564:Membrane Potentials; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D000609:Aminoacridines; D007555:Isoxazoles; D008051:Lipid Bilayers; C010847:9-amino-6-chloro-2-methoxyacridine; C031184:oxonol VI; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases
keywords: Membrane potential; Proteoliposome; Proton pumping; pH-sensitive dye