pH dependence of H+ conduction through the membrane moiety of the H+-ATPase (F0 . F1) and effects of tyrosyl residue modification.
A convenient and reliable method to measure passive H+-translocating activity (H+ conductivity) was developed; vesicles reconstituted from the membrane moiety (F0) of H+-ATPase (F0 . F1) and soybean phospholipids were loaded with KCl by a freeze-thaw-sonication procedure and the rate of H+ uptake caused by the K+ diffusion potential upon addition of valinomycin was followed with a pH meter. Of the methods tested, a dialysis method using cholate plus deoxycholate gave the best results for reconstitution. Using this method, H+ conductivity of the membrane moiety of H+-ATPase from a thermophilic bacterium PS3 (TF0) was analyzed. Dependence of H+ conductivity of TF0 on H+ concentration fitted a Michaelis-Menten equation showing a Vmax of 31.3 microgram ion/min . mg of TF0 and a Km of 0.095 microgram ion/liter. Upon modification of a tyrosyl residue of TF0 with iodine, the Km value shifted to 0.71 microgram ion/liter, while the Vmax remained constant. These results were interpreted as indicating that a single tyrosyl residue in N,N'-dicyclohexylcarbodiimide-binding proteolipid of TF0 plays an important role as an H+ donor in the the rate-limiting step of H+ permeation through TF0. TF1, the catalytic moiety of H+-ATPase from the thermophilic bacterium PS3, blocked H+ conduction through TF0. A 1:1 stoichiometry of TF1 and TF0 was found in ATP-dependent membrane potential generation as well as H+ conduction.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D010743:Phospholipids / Q000494:pharmacology; D006180:Proton-Translocating ATPases; D014443:Tyrosine; D014634:Valinomycin / Q000494:pharmacology
D010743:Phospholipids; D014634:Valinomycin; D014443:Tyrosine; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases