Anion-Sensitive, H+-Pumping ATPase in Membrane Vesicles from Oat Roots

Abstract

H(+)-pumping ATPases were detected in microsomal vesicles of oat (Avena sativa L. var Lang) roots using [(14)C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (DeltapH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K(+)), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H(+) pumping as measured by MeA uptake were characterized. The K(m) (app) for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N'-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl(-) > Br(-) > NO(3) (-) > SO(4) (2-), iminodiacetate, benzene sulfonate. Anion stimulation of H(+) pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl(-) by a H(+) -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K(+) in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. DeltapH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H(+) -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H(+) pumps at the plasma membrane and tonoplast of higher plant cells.