Reaction kinetics of the vacuolar H(+)-pumping ATPase in beta vulgaris.
Vacuolar-type H(+)-ATPases (V-ATPases) are ubiquitous in eukaryote endomembranes, where they are responsible for lumenal acidification. The ratios of H+ translocated per ATP hydrolyzed (which may be important in controlling the activity of these pumps) have previously been found to be variable, noninteger and sensitive to cytosolic as well as lumenal pH. The mechanistic implications of these findings are explored here with reaction kinetics. Experimental data for this analysis comprise supra- and superlinear V-ATPase current-voltage relationships, isolated as bafilomycin-sensitive currents in vacuolar membranes from Beta using the "whole vacuole" patch clamp configuration. Whereas simple models with one reaction cycle fail to provide an adequate description of the data (mainly because of a weak sensitivity of the zero-current voltage to the transmembrane pH gradient), a model with two linked reaction loops allowing partial coupling of H+ translocation to ATP hydrolysis does provide good descriptions. All experimental data obtained with the same vacuolar pH (4.3) could be reduced to a model with eleven independent parameters. Best fits have been obtained on the basis of a binding domain possessing 3 H+ binding sites per ATP hydrolyzed and a net charge of -2 when unoccupied. The enzyme could reorientate its access site between the vacuolar and cytoplasmic side when zero, one or three H+ are bound.
Davies
J M
JM
Department of Biology, University of York, United Kingdom.
Sanders
D
D
Gradmann
D
D
D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000502:physiology*; D007700:Kinetics; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D018521:Plant Physiological Phenomena; D010944:Plants / Q000648:ultrastructure; D017494:Proton Pumps / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013816:Thermodynamics; D014617:Vacuoles / Q000502:physiology*
D017494:Proton Pumps; D006180:Proton-Translocating ATPases