H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli.
A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound [H+]-ATPases. In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient. At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate. This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry. The method has been applied to two different [H+]-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group). The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells. In contrast, the E. coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.
D000165:Acridine Orange; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011522:Protons; D013050:Spectrometry, Fluorescence
D007473:Ion Channels; D011522:Protons; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000165:Acridine Orange