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 reconst ituted into artificial proteoliposomes, During purification, a vanadat e- and nitrate-sensitive ATPase activity, consisting of partially disa ssembled V-ATPase complexes, was resolved from the V-ATPase peak, ATPa se and H+-transport activities of the purified, reconstituted V-ATPase s of both fruit and epicotyl exhibited similar inhibitor profiles, exc ept that the fruit V-ATPase retained partial vanadate sensitivity, Sin ce the V-ATPase activity of native fruit tonoplast vesicles is insensi tive 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 t he reconstituted epicotyl V-ATPase exhibited twice as much intrinsic u ncoupling or slip as the reconstituted fruit V-ATPase, Comparison of t heir subunit compositions by SDS polyacrylamide gel electrophoresis in dicated that the reconstituted fruit V-ATPase is enriched in two polyp eptides of 33/34 and 16 kDa, Moreover, the stalks of negatively staine d juice sac V-ATPases appeared thicker than those of epicotyl V-ATPase s in electron micrographs.