Anion-Sensitive, H+-Pumping ATPase of Oat Roots

Abstract

To understand the mechanism and molecular properties of the tonoplast-type H(+)-translocating ATPase, we have studied the effect of Cl(-), NO(3) (-), and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H(+)-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H(+)-pumping ATPase generates a membrane potential (Deltapsi) and a pH gradient (DeltapH) that make up two interconvertible components of the proton electrochemical gradient (Deltamuh(+)). A permeant anion (e.g. Cl(-)), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([(14)C]thiocyanate distribution) and stimulated formation of a pH gradient ([(14)C]methylamine distribution). However, Cl(-)-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl(-) independent of the proton electrochemical gradient. Unlike the plasma membrane H(+)-ATPase, the Cl(-)-stimulated ATPase was inhibited by NO(3) (-) (a permeant anion) and by DIDS. In the absence of Cl(-), NO(3) (-) decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO(3) (-) of Cl(-)-stimulated pH gradient formation and Cl(-)-stimulated ATPase activity was noncompetitive. In the absence of Cl(-), DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl(-)-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H(+)-ATPase by NO(3) (-) or DIDS suggest that the vanadate-insensitive H(+)-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H(+)-ATPase has channels that conduct anions is yet to be established.