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.

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Anion-Sensitive, H<sup>+</sup>-Pumping ATPase of Oat Roots <sup><a href="#fn1" rid="fn1" class=" fn">1</a></sup>

K Churchill

H Sze

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 (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (} $\bar{Δ}$ μh^{). A permeant anion (}e.g. Cl^{), unlike an impermeant anion (}e.g. iminodiacetate), dissipated the membrane potential ([^{C]thiocyanate distribution) and stimulated formation of a pH gradient ([}^{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.}

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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

^{Present address: Biology Department, Yale University, New Haven, CT 06511.}

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

Copyright notice

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 (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (}

\bar{Δ}

μh^{). A permeant anion (}e.g. Cl^{), unlike an impermeant anion (}e.g. iminodiacetate), dissipated the membrane potential ([^{C]thiocyanate distribution) and stimulated formation of a pH gradient ([}^{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.}

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