Properties of the tonoplast ATP-dependent H + pump of Acer pseudoplatanus: inhibition by nitrate and vanadate

Abstract

Sealed tonoplast vesicles were prepared from vacuoles of Acer pseudoplatanus cells. The rate of contamination by plasmalemma never exceeded 3%. The vesicles of tonoplast exhibited ATP- and PP i-dependent proton translocation activity. The properties of ATP-dependent H +-transport were studied and compared to those of ATP hydrolysis previously described (A. Pugin et al., in: B. Marin (Ed.), Plant Vacuoles, NATO ASI Series, Serie A, Vol. 134, Plenum Press, 1987, pp. 135–141). H +-transport and enzymatic properties of the tonoplast ATPase were well correlated. The ATP-dependent H +-translocation into the vesicles had an optimum around pH 7.3; MgATP 2− was preferentially used as a substrate with an apparent K m of 153 μM. ATP-dependent H + transport was stimulated by anions. This effect was shown to be partly due to the ability of permeant anions to dissipate the electrical potential Δψ. When Δψ was collapsed, H +-ATPase activity was stimulated by Cl − and inhibited by NO 3 −. The use of three pH probes: acridine orange (AO), 9-amino-6-chloro-2-methoxyacridine (ACMA) and quinacrine confirmed the limits of acridine orange for the measurement of pH gradients in the presence of NO 3 −. Various chemicals were potent inhibitors of the tonoplast H +-ATPase, the sulfhydryl reagents N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), mersalyl, the lipid soluble carbodiimide N, N′-dicyclohexylcarbodiimide (DCCD), the synthetic estrogen diethylstilbestrol (DES) and vanadate which is often reported as a specific inhibitor of the plasmalemma ATPase. The inefficiency of vanadate on the tonoplastic PP i-dependent H + transport indicated that the inhibition of the ATP-dependent H +-pump resulted from an effect on the enzyme. The sensitivity of the tonoplastic H +-ATPase to vanadate is discussed. We suggest that there are V-type ATPases which could operate via a phosphoenzyme intermediate.