Reconstitution of H+ATPase into Planar Phospholipid Bilayers and Its Kinetic Analysis
The proton-translocating ATPase of the thermophilic bacterium PS3 (TFoF1) was incorporated into planar phospholipid bilayers, and its electrogenicity and kinetic characteristics were examined. A short-circuit current of up to 1 nA/cm2 was generated upon the addition of ATP. The generation of the electric current was progressively suppressed by inhibitors of TF1. From the reversal potential, the electrogenicity of TFoF1 was indicated to be some 180 mV. The relationship between the electric current induced by ATP and the concentration of ATP revealed that the magnitude of the electric current followed simple Michaelis-Menten type kinetics and the Km was found to be 0.14 mM under the conditions studied. Furthermore, there was no apparent dependence of the Km on externally applied membrane potential. These results suggest that the voltage dependence resides in some steps that defines the apparent Vmax rather than Km in the reaction cycle.
KeywordsElectric Current Voltage Dependence Steady State Current Electric Capacitance Proton Translocation
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