Characterization of a Proton-Translocating ATPase in Microsomal Vesicles from Corn Roots (original) (raw)

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg2-ATPase activity of the sealed vesicles was stimulated by Cl-and NHIt and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The lonophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 65. The ATPase was inhibited by N03-, was insensitive to K+, and was not inhibited by 100 micromolar vanadate or by 1 milfimlar azide. Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg2+, was stimulated by ClI, inhibited by N03-, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATPdependent fluorescence quench both required a divalent cation (MgNW 2 Mn2" > Co2") and were inhibited by high concentrations of Ca2". The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the protontranslocating ATPase differed from those of the mitochondrial F1F,-ATPase and from those of the K+-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase. ATP-dependent proton transport and ATP-dependent generation of membrane potentials have been demonstrated in microsomal vesicles obtained from several plant tissues. Methylamine uptake (16), imidazole uptake (22), shift in wavelength of neutral red dye (9, 10), and quenching of fluorescence of quinacrine (3, 6, 7), 9-amino acridine (3), and acridine orange (26) have all been used to demonstrate ATP-dependent acidification of the interior of microsomal vesicles from higher plants. SCN-uptake (19, 22, 25) and shift in absorbance spectrum of the membrane-potentialsensitive dye oxanol (3) have been used to demonstrate generation of a positive membrane potential, discharged by protonophores, in the microsomal vesicles. The ATPase activity of the vesicles is stimulated by ionophores (3, 6, 7, 20, 21, 23-25). Thus, there is now excellent evidence for the presence of an electrogenic, protontranslocating ATPase which utilizes externally supplied ATP to 'Supported by Grant PCM 78-12119 from the National Science Foundation to R. M. Spanswick.