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(Received for publication, October 24, 1996, and in revised form, February 10, 1997)
From the Biology Department, Sinsheimer Laboratories, University of
California, Santa Cruz, California 95064
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.
Volume 272, Number 19,
Issue of May 9, 1997
pp. 12762-12770
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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