Plant Physiology 71:610-617 (1983)
© 1983 American Society of Plant Biologists
Articles
Anion-Sensitive, H+-Pumping ATPase in Membrane Vesicles from Oat Roots 1
Kathleen A. Churchill and
Heven Sze
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
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 ( pH) (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 Kmapp 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. pH 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.
1 Supported in part by the General Research Fund of the University of Kansas and National Science Foundation Grant PCM 80-16808 to H. S. and a Sigma Xi Grant-in-Aid of Research to K. A. C.
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