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

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 PPi-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; MgATP2− was preferentially used as a substrate with an apparent Km 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 NO3−. 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 NO3−. 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 PPi-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.