Steady State Kinetics of Proton Translocation Catalyzed by Thermophilic F0F1-ATPase Reconstituted in Planar Bilayer Membranes

The proton-translocating ATPase of the thermophilic bacterium PS3 was reconstituted into planar phospholipid bilayers by the previously reported method (Hirata, H., Ohno, K., Sone, N., Kagawa, Y., and Hamamoto, T. (1986) J. Biol. Chem. 261, 9839–9843), and the relationship between the electric curre...

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Veröffentlicht in:The Journal of biological chemistry 1989-04, Vol.264 (11), p.6092-6096
Hauptverfasser: Muneyuki, E, Kagawa, Y, Hirata, H
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creator Muneyuki, E
Kagawa, Y
Hirata, H
description The proton-translocating ATPase of the thermophilic bacterium PS3 was reconstituted into planar phospholipid bilayers by the previously reported method (Hirata, H., Ohno, K., Sone, N., Kagawa, Y., and Hamamoto, T. (1986) J. Biol. Chem. 261, 9839–9843), and the relationship between the electric current induced by ATP and the concentration of ATP was examined. The magnitude of the electric current generated upon addition of ATP followed simple Michaelis-Menten type kinetics, and the Michaelis constant was found to be 0.14 mM under our conditions. This value is close to the values reported for F1- or F0F1-ATPase in its steady state catalytic cycle, indicating that the proton translocation is coupled to the steady state ATPase reaction. The relationship between the Km value and the membrane potential was also examined under the voltage-clamped condition, and we found that there was no apparent dependence of the Km on membrane voltage. These results together with the previous data suggest that the voltage dependence residues in some step that defines the apparent Vmax rather than Km in the reaction cycle, and proton translocation is not directly coupled to this ATP binding step.
doi_str_mv 10.1016/S0021-9258(18)83317-8
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(1986) J. Biol. Chem. 261, 9839–9843), and the relationship between the electric current induced by ATP and the concentration of ATP was examined. The magnitude of the electric current generated upon addition of ATP followed simple Michaelis-Menten type kinetics, and the Michaelis constant was found to be 0.14 mM under our conditions. This value is close to the values reported for F1- or F0F1-ATPase in its steady state catalytic cycle, indicating that the proton translocation is coupled to the steady state ATPase reaction. The relationship between the Km value and the membrane potential was also examined under the voltage-clamped condition, and we found that there was no apparent dependence of the Km on membrane voltage. 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(1986) J. Biol. Chem. 261, 9839–9843), and the relationship between the electric current induced by ATP and the concentration of ATP was examined. The magnitude of the electric current generated upon addition of ATP followed simple Michaelis-Menten type kinetics, and the Michaelis constant was found to be 0.14 mM under our conditions. This value is close to the values reported for F1- or F0F1-ATPase in its steady state catalytic cycle, indicating that the proton translocation is coupled to the steady state ATPase reaction. The relationship between the Km value and the membrane potential was also examined under the voltage-clamped condition, and we found that there was no apparent dependence of the Km on membrane voltage. 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source MEDLINE; Alma/SFX Local Collection; EZB Electronic Journals Library
subjects Adenosine Triphosphate - metabolism
Artificial membranes and reconstituted systems
Bacteria - enzymology
Biological and medical sciences
Biological Transport, Active
Electric Conductivity
Fundamental and applied biological sciences. Psychology
Hot Temperature
Hydrogen-Ion Concentration
In Vitro Techniques
Kinetics
Lipid Bilayers
Membrane physicochemistry
Membrane Potentials
Molecular biophysics
Proton-Translocating ATPases - metabolism
title Steady State Kinetics of Proton Translocation Catalyzed by Thermophilic F0F1-ATPase Reconstituted in Planar Bilayer Membranes
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