Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATP-driven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes were less permeable to protons than epicotyl membranes. H pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N`-dicyclohexylcarbodiimide. In addition, the epicotyl H pumping activity was inactivated by oxidation at room temperature, and oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate (100 mM) was correlated with the release of V complexes from the membrane. In contrast, H pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the V-ATPase inhibitors, and resistant to oxidation. Unlike the epicotyl H-ATPase, the fruit H-ATPase activity was partially inhibited by 200 µM vanadate. Cold inactivation treatment failed to inhibit H pumping activity of the fruit membranes, even though immunoblots showed that V complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 µM vanadate from 30% to 60%. These results suggest the presence of two H-ATPases in the fruit preparation: a V-ATPase and an unidentified vanadate-sensitive H-ATPase. Attempts to separate the two activities in their native membranes on linear sucrose density gradients were unsuccessful. However, following detergent-solubilization and centrifugation on a glycerol density gradient, the two ATPase activities were resolved: a nitrate-sensitive V-type ATPase that is also partially inhibited by 200 µM vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.

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