ON THE MECHANISM OF HYPERACIDIFICATION IN LEMON - COMPARISON OF THE VACUOLAR H-ATPASE ACTIVITIES OF FRUITS AND EPICOTYLS()

Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidificat ion, the kinetics of ATP-driven proton pumping by tonoplast vesicles f rom 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 nitr ate, bafilomycin, N-ethylmaleimide, and N,N'-dicyclohexylcarbodiimide. In addition, the epicotyl H+ pumping activity was inactivated by oxid ation at room temperature, and oxidation was reversed by dithiothreito l. Cold inactivation of the epicotyl V-ATPase by nitrate (greater than or equal to 100 mM) was correlated with the release of V-1 complexes from the membrane. In contrast, H+ pumping by the fruit tonoplast-enri ched 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 mu M vanadate. Cold inactivation treatment failed to inhibit H+ pumping activity of the fruit membranes, even though immunoblots showed that V -1 complexes were released from the membrane. However, cold inactivati on doubled the percent inhibition by 200 mu M vanadate from 30% to 60% . These results suggest the presence of two H+-ATPases in the fruit pr eparation: 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, followin g detergent-solubilization and centrifugation on a glycerol density gr adient, the two ATPase activities were resolved: a nitrate-sensitive V -type ATPase that is also partially inhibited by 200 mu M vanadate, an d an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.