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Electrogenic Transport of Protons Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish ¹

Biochemical Characterization

Centro di Studio del C.N.R. per la Biologia Cellulare e Molecolare delle Piante, Università di Milano, via G. Celoria 26, 20133 Milano, Italy, Dipartimento di Biologia `L. Gorini', Università di Milano, via G. Celoria 26, 20133 Milano, Italy

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference ( ). pH was measured as the decrease of absorbance of Acridine orange and as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent pH and generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, pH generation is not inhibited by NO₃^–. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and generation are influenced by anions: pH increases and decreases following the sequence SO₄^2–, Cl^–, Br^–, NO₃^–. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. pH generation is specificially 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.