A H-translocating inorganic pyrophosphatase (H -PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H'-PPase catalyzed the electrogenic transport of H' into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb' = K' > Cs'; but it was inhibited by Na' or Li'. Maximum rates of transport required at least 20 millimolar K' and the K. for this ion was 4 millimolar. Fluoride inhibited both ApH formation and K'-dependent PPase activity with an I50 of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H'-ATPase (e.g. a disulfonic stilbene or NO3-) had no effect on the PPase activity. Vanadate and azide were also ineffective. HW-pumping PPase was inhibited by 7-chloro4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N'-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H'-PPase and the H'-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H 'Abbreviations: PPase, inorganic pyrophosphatase; BTP, bis-tris-propane or 1 ,3-bis[tris(hydroxymethyl)methylamine]propane; DCCD, N,N'-dicyclohexylcarbodiimide; DIDS, 4,4'-diisothiocyanostilbene-2,2'disulfonate; NBD-CI. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; NEM, N-ethylmaleimide; ApH, pH gradient; A#, membrane potential; IDA, iminodiacetate; Oxonol V, bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol.that it might be a H+ pump. Churchill and Sze (6) tested this idea with a low density vesicle preparation from oat roots enriched in tonoplast vesicles; and found that PPi generated a pH gradient in these vesicles. More recent studies by Rea and Poole (19) and Chanson el al. (5) have shown that the PPase5 of the low density vesicles from red beets and corn coleoptiles is a H+ pump. Their work further indicates that the PPase is a separate enzyme and not a partial activity of the H+-pumping ATPase that is associated with the tonoplast. However, the properties of the PPase and its physiological function are still poorly understood.Here we show the H+-pumping PPase from oat roots is electrogenic and sensitive to fluoride inhibition. Under certain conditions, the ApH generated by the H+-PPase is similar in magnitude to that generated at optimal conditions by the H+-ATPase. A preliminary report of these results has been presented (29).MATERIALS AND METHODS Plant Material. Oats (Avena sativa L. var. Lang) were germinated in the dark over an aerated solution of 0.5 mm CaSO4. After 4 or 5 d of growth, the apical sections (3-4 cm) of the roots were harvested.Preparation of Low Density Vesicles. The procedure of Churchill and Sze (6, 7) was used with some modifications. Roots were homogenized twice with a mortar and pestle in medium containing...