Purification and reconstitution of the vacuolar H+-ATPases from lemon fruits and epicotyls.
The vacuolar H+-ATPases (V-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 (Müller, 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.
Müller
M L
ML
Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064, USA.
Irkens-Kiesecker
U
U
Kramer
D
D
Taiz
L
L
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005638:Fruit / Q000201:enzymology*; D008854:Microscopy, Electron; D008970:Molecular Weight; D017494:Proton Pumps / Q000302:isolation & purification* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases
D017494:Proton Pumps; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases