Electrogenic Transport of Protons Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish

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Abstract
Mg:ATP-dependent H+ pumping was studied in microsomal vesicles from 24 h old radish seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (.DELTA. .psi.). .DELTA. pH was measured as the decrease of absorbance of acridine orange and .DELTA. .psi. as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent .DELTA. pH and .DELTA. .psi. generation are completely inhibited by vanadate and insensitive to oligomycin; .DELTA. pH generation is not inhibited by NO3-. This membrane preparation apparently is virtually devoid of mitochondrial and tonoplast H+-ATPases. Both intravesicular acidification and .DELTA. .psi. generation are influenced by anions: .DELTA. pH increases and .DELTA. .psi. decreases following the sequence SO42-, Cl-, Br-, NO3-, ATP-dependent H- pumping strictly requires Mg2+. It is very specific for ATP (apparent Km 0.76 mM) compared to GTP, UTP, CTP or ITP. .DELTA. pH generation is inhibited by CuSO4 and diethylstilbestrol as well as vanadate. .DELTA. pH generation is specifically stimulated by K+ (+80%) and to a lesser extent by Na+ and choline (+28% and +14%, respectively). The characteristics of H+ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.