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 [¹⁴C]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 (ΔpH) (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_mapp 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⁻ > NO₃⁻ > SO₄²⁻, 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. ΔpH 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.