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Anion-Sensitive, H⁺-Pumping ATPase of Oat Roots ¹

Direct Effects of Cl^–, NO₃^–, and a Disulfonic Stilbene

Department of Botany, University of Kansas, Lawrence, Kansas 66045, Department of Biochemistry, University of Kansas, Lawrence, Kansas 66045, Department of Botany, University of Maryland, College Park, Maryland 20742

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied 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 (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential () and a pH gradient (pH) that make up two interconvertible components of the proton electrochemical gradient (µH⁺). 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). 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 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-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.

¹ Supported in part by National Science Foundation Grants PCM 83-10928 and PCM 83-04130 to H. S.

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