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

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Abstract
To understand the mechanism and molecular properties of the tonoplast-type H+-translocating ATPase, the effect of Cl-, NO3- 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 was studied. The H+-pumping ATPase generates a membrane potential (.DELTA..psi.) and a pH gradient (.DELTA.pH) that make up 2 interconvertible components of the H+ electrochemical gradient (.DELTA..hivin..mu.H+). A permeant anion (e.g., Cl-), unlike an impermeant anion (e.g., iminodiacetate), dissipated the membrane potential ([14C]thiocyanate distribution) and stimulated formation of a pH gradient ([14C]methylamine distribution). Cl--stimulated ATPase activity was .apprx. 75% caused by a direct stimulation of the ATPase by Cl- independent of the H+ electrochemical gradient. Unlike the plasma membrane H+-ATPase, the Cl--stimulated ATPase was inhibited by NO3- (a permeant anion) and by DIDS. In the absence of Cl-, NO3- decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO3- 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 NO3- 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-selective H+-ATPase has channels that conduct anions is yet to be established.