title: Steady state kinetics of proton translocation catalyzed by thermophilic F0F1-ATPase reconstituted in planar bilayer membranes.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Faculty of Science, University of Tokyo, Japan.
authors.forename: E
authors.initials: E
authors.lastname: Muneyuki
authors.forename: Y
authors.initials: Y
authors.lastname: Kagawa
authors.forename: H
authors.initials: H
authors.lastname: Hirata
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology; D001693:Biological Transport, Active; D004553:Electric Conductivity; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials; D006180:Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases