Purification and reconstitution of the vacuolar H+-ATPases from lemon fruits and epicotyls

The vacuolar H+-ATPases CV-ATPases) of lemon fruits and epicotyls were detergent-solubilized, purified by column chromatography, and reconstituted into artificial proteoliposomes, During purification, a vanadate- and nitrate-sensitive ATPase activity, consisting of partially disassembled V-ATPase complexes, was resolved from the V-ATPase peak, ATPase and H+-transport activities of the purified, reconstituted V-ATPases of both fruit and epicotyl exhibited similar inhibitor profiles, except that the fruit V-ATPase retained partial vanadate sensitivity, Since the V-ATPase activity of native fruit tonoplast vesicles is insensitive to inhibitors (Muller, M, L,, Irkens-Kiesecker, U,, Rubinstein, B,, and Taiz, L. (1996) J, Biol, Chem, 271, 1916-1924), membrane lipids or other factors may protect the fruit V-ATPase from inactivation in vivo. A kinetic analysis of H+-pumping and H+-leakage indicated that the reconstituted epicotyl V-ATPase exhibited twice as much intrinsic uncoupling or slip as the reconstituted fruit V-ATPase, Comparison of their subunit compositions by SDS polyacrylamide gel electrophoresis indicated that the reconstituted fruit V-ATPase is enriched in two polypeptides of 33/34 and 16 kDa, Moreover, the stalks of negatively stained juice sac V-ATPases appeared thicker than those of epicotyl V-ATPases in electron micrographs.