From the Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064
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ABSTRACT |
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Lemon fruit tonoplasts, unlike those of seedling
epicotyls, contain nitrate-insensitive H+-ATPase
activity (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916-1924). However, the degree of nitrate-insensitivity fluctuates
during the course of the year with a seasonal frequency. Nitrate
uncouples H+ pumping from ATP hydrolysis both in epicotyls
and in nitrate-sensitive fruit V-ATPases. Neither bafilomycin nor
oxidation cause uncoupling. The initial rate H+/ATP
coupling ratios of epicotyl and the nitrate-sensitive fruit proton
pumping activities are the same. However, the H+/ATP
coupling ratio of the nitrate-insensitive fruit H+ pumping
activity is lower than that of nitrate-sensitive and epicotyl
V-ATPases. Several properties of the nitrate-insensitive H+-ATPase of the fruit indicate that it is a modified
V-ATPase rather than a P-ATPase: 1) insensitivity to low concentrations
of vanadate; 2) it is initially strongly uncoupled by nitrate, but
regains coupling as catalysis proceeds; 3) both the nitrate-sensitive and nitrate-insensitive fruit H+-pumps have identical
Km values for MgATP, and show similar pH-dependent slip and proton leakage rates. We conclude
that the ability of the juice sac V-ATPase to build up steep pH
gradients involves three factors: variable coupling,
i.e. the ability to regain coupling under conditions that
initially induce uncoupling; a low pH-dependent slip rate;
the low proton permeability of the membrane.
The vacuolar H+-ATPases
(V-ATPases)1 of eukaryotic
cells are large, multimeric proton pumps composed of 10 to 13 different
subunits organized into a hydrophilic catalytic complex,
V1, and a hydrophobic transmembrane H+ channel,
Vo (1). V-ATPases are structurally related to the ATP
synthases, or F-ATPases, of mitochondria and chloroplasts. F- and
V-ATPases exhibit many structural and functional similarities at
the molecular level, including several homologous subunits which play
central roles in catalysis and proton transport (2, 3). Because of
these similarities in structure and catalytic mechanism, F- and
V-ATPases are also thought to exhibit similar mechanochemical
properties (4). When operating in ATP synthesis mode, F-ATPases convert
the proton-motive force present across the internal membrane of
chloroplasts and mitochondria into a rotary torque used to drive the
synthesis of ATP by the catalytic F1 sector (5, 6).
Conversely, when F-ATPases operate in proton pumping mode, they
hydrolyze ATP at their catalytic site and convert the liberated energy
into a rotary torque utilized to drive proton transport across the
membrane (7). V-ATPases are also thought to operate via a rotary
mechanism, even though direct experimental evidence for rotation is
lacking (1).
In V- and F-ATPases, the efficiency of coupling between ATP hydrolysis
and proton transport represents a critical, rate-limiting factor. Both
pumps have been proposed to undergo slip, or intrinsic uncoupling,
under specific conditions (8-10). During slip, ATP is hydrolyzed at
the catalytic site without being coupled to H+ transport.
Since typical V-ATPases are known to operate far from thermodynamic
equilibrium, slip may represent one of the enzyme's regulatory mechanisms.
In lemon fruit juice sac cells, the vacuolar pH can reach as low as
2.2, about 3 pH units lower than in typical plant vacuoles. In fact,
the trans-tonoplast pH gradient in lemon fruits is close to the
calculated maximum for a V-ATPase operating at thermodynamic equilibrium (4 to 5 pH units) assuming an H+/ATP
stoichiometry of 2 (11, 12). We have previously shown that proton
pumping by tonoplast-enriched juice sac vesicles is largely insensitive
to the V-ATPase inhibitors nitrate, bafilomycin A1, and
partially sensitive to high vanadate concentrations (50 to 300 µM) (13). The proton pumping activity of juice sac
vesicles is also less sensitive to oxidation and
N-ethylmaleimide (NEM) than that of tonoplast-enriched
vesicles from seedling epicotyls. In addition, cold inactivation in the
presence of nitrate, which completely inhibits proton transport in
epicotyl vesicles, has little effect on the H+ pumping
activity of juice sac vesicles, even though the treatment induces the
release of similar proportions of catalytic subunits from both types of
vesicles. Since V1 dissociation treatment increased the
sensitivity of the juice sac H+ pumping activity to
vanadate, we hypothesized that a second, vanadate-sensitive proton
pump, possibly possessing a lower H+/ATP stoichiometry that
enables it to generate a steeper pH gradient than the V-ATPase, may be
present on the tonoplasts of juice sac cells (13).
After purification and reconstitution into artificial proteoliposomes,
both the epicotyl and juice sac proton pumps exhibited equal
sensitivities to nitrate, bafilomycin A1, NEM, and
oxidation. Thus, the insensitivity of the juice sac H+
pumping activity to these inhibitors appears to depend on some component(s) of the native membrane. However, purified and
reconstituted fruit V-ATPases remained partially sensitive to vanadate
and exhibited only half as much slip as the epicotyl V-ATPase (14).
Slip was calculated from proton pumping curves, in the absence of an
electric potential gradient, by the method of Tu et al.
(15). Under these conditions, slip reflects intrinsic uncoupling
induced by the build-up of a pH gradient across the membrane. This
method gives no indication on the H+/ATP coupling ratio of
the pump in the absence of a pH gradient.
H+/ATP coupling ratios provide a measure of pump efficiency
(16). If two pumps have the same H+/ATP stoichiometry, but
differ in their coupling ratios, the pump with the higher
H+/ATP coupling ratio would be expected to generate a
steeper A 20 month analysis of the nitrate sensitivity of tonoplast-enriched
vesicles from juice sacs showed that the proportion of nitrate
resistant activity varied during the course of the year. As the
proportion of uncoupled enzymes varies, the overall H+/ATP
coupling ratio measured in juice sac vesicles changes. The significance
of this type of variable H+/ATP coupling in lemon fruits is
unclear, but the nitrate resistant activity may represent a specialized
group of V-ATPases which, under extreme conditions, is able to
maintain, if not build up, the large Materials--
Lemon seeds (Citrus limon L. var.
Schaub Rough Lemon) for growing seedling epicotyls were generously
supplied by Willits & Newcomb, Inc., Arvin, CA. Lemon fruits (var.
Eureka) were harvested from a tree on the campus of the University of
California, Santa Cruz. Reduced nicotinamide-adenine dinucleotide
(NADH) was from Boehringer Mannheim. All other chemicals were purchased
from Sigma or Fisher.
Membrane Preparation--
Tonoplast-enriched membranes from
lemon fruit juice sacs and epicotyls were prepared as described
previously (14). Fruit juice sacs were homogenized in fruit
homogenization buffer (1.5 M MOPS-KOH, pH 8.5, 2.25%
polyvinylpyrrolidone 40, 0.75% bovine serum albumin, 7.5 mM EDTA, 2 mM DTT, and 0.1 mM
phenylmethylsulfonyl fluoride) and epicotyls were homogenized in
epicotyl homogenization buffer (0.5 M MOPS-KOH, pH 8.5, 1.5% polyvinylpyrrolidone 40, 0.5% bovine serum albumin, 5 mM EDTA, 2 mM DTT, and 0.1 mM
phenylmethylsulfonyl fluoride). After a first centrifugation of the
homogenates for 20 min at 12,000 × g, the supernatants
was subjected to ultracentrifugation for 60 min at 132,000 × g, and the microsomal pellets obtained were further purified
on a 10/35% sucrose step gradient for 60 min at 132,000 × g. The 10/35% interface containing tonoplast-enriched membranes was recovered, diluted with RB (10 mM BTP-Mes, pH
7.0, 20 mM KCl, 1 mM EDTA, 2 mM
DTT, and 0.1 mM phenylmethylsulfonyl fluoride), and
pelleted for 20 min at 174,000 × g. The
tonoplast-enriched membranes were resuspended in RB at a final
concentration of ~5 µg of membrane protein/µl.
V1 Dissociation--
Membrane vesicles were made up
to 0.3 mg of protein/ml in 2.5 ml of RB containing 5 mM
ATP, 7 mM MgSO4, and 0 or 500 mM
KNO3. They were incubated on ice for 1 h and
centrifuged 15 min at 412,000 × g. The membrane pellet
was resuspended in 750 µl of RB and used for activity measurements
and immunoblotting. The proteins in 50 µl of the resuspended pellet
and 167 µl of the supernatant were used for immunoblotting after
being precipitated with 10% trichloroacetic acid, washed with cold
acetone, lyophilized, and separated by SDS-PAGE.
Activity Assays--
Proton pumping and ATP hydrolysis by
tonoplast-enriched vesicles were measured simultaneously by using the
continuous spectrophotometric assay of Palmgren (17). In this assay,
proton transport into the vesicles was followed by measuring the
absorbance decrease of acridine orange at 495 nm. Simultaneously, ATP
hydrolysis was measured by coupling the appearance of ADP to the
oxidation of NADH and by following the NADH absorbance decrease at 340 nm. The assay medium consisted of 10 mM MOPS-BTP, pH 7.0, 2 mM ATP, 150 mM KCl, 1 mM sodium
azide, 0.5 µM valinomycin, 0.25 mM very freshly prepared NADH, 1 mM phosphoenolpyruvate, 20 µM acridine orange, and 25 µl/ml of a mixture of
pyruvate kinase and lactate dehydrogenase (Sigma). 50 µM
sodium vanadate was also present in the mixture unless stated
differently. The reaction was started with 4 mM
MgCl2 and the absorbance values at 340 and 495 nm were recorded in 15-s intervals with a Spectronic Genesis 5 spectrophotometer (Spectronic, Rochester, NY) interfaced with a PC
running the MultiWL computer program (M. L. Müller and A. Murphy). After correction for mixing artifacts, the results were loaded
into a graphics program and plotted out (Fig.
1). When activity rates are reported, the
slopes of the initial rates of proton pumping and ATP hydrolysis were
calculated and expressed in arbitrary units (a.u.). The calculated H+/ATP coupling ratios were independent of the amount of
membrane protein used in the assay (data not shown). We nevertheless
chose to do all experiments in the presence of 20-25 µg of membrane protein, unless stated differently. When proton pumping was measured in
the presence of nitrate in the reaction mixture, quinacrine fluorescence quenching was substituted for acridine orange absorbance quenching. If the effect of nitrate was to be assessed on proton pumping alone, the conditions were as described previously (13). If
H+/ATP coupling ratios were to be determined, quinacrine
fluorescence quenching was measured in the assay mixture described
above, so that H+ pumping and ATP hydrolysis activities
were measured in the same conditions. Because the initial rate of
H+ pumping differed from the maximum activity rate in the
presence of nitrate, the latter was chosen for all activity
measurements in the presence of nitrate.
Immunoblotting--
The proteins of tonoplast-enriched vesicles
from epicotyls and juice sacs were separated by SDS-PAGE according to
Laemmli (18) in 12% polyacrylamide gels and the transfer to
nitrocellulose was done as described previously (13). The blots were
incubated with a primary antibody to the 70-kDa subunit of the corn
V-ATPase and visualized by a peroxidase-coupled secondary antibody
reaction (Vectastain ABC kit, Vector Laboratories, Burlingame, CA)
(13).
Protein Concentration--
Estimates of protein concentrations
were done routinely with Amido Black (19).
Sensitivity to Nitrate--
Although we had previously found that
proton pumping by fruit juice sac vesicles was largely insensitive to
inhibition by nitrate and bafilomycin A1 (13) (Table
I), we observed that there was some
variability in the nitrate insensitivity
of the lemon fruit V-ATPase from one membrane preparation to another (Fig. 2). Fig. 3A shows the
nitrate sensitivity of tonoplast-enriched fruit vesicles from
preparations made over a period of 20 months. All fruits were harvested
at the same state of development from a single tree situated on the
campus of the University of California, Santa Cruz. We found that, over
the duration of the experiment, the inhibition of the proton pumping
activity by 100 mM KNO3 followed a sigmoidal
pattern, ranging from 0 to 20% inhibition in the fall to 70-80% in
the spring.
In addition to the seasonal variation in nitrate sensitivity noted
above, a temporal component was also observed in the kinetic study of
nitrate inhibition. As shown in Fig. 3B, even in a
"nitrate-insensitive" juice sac preparation, H+ pumping
appeared to be initially inhibited by nitrate. However, after 1-2 min,
the activity underwent a progressive "recovery" to reach a maximum
rate comparable to that of the control. This temporal delay in the
attainment of the maximum rate of proton pumping was observed under all
experimental conditions used (including experiments done in the
presence of acridine orange) and whether an ATP regeneration system was
present or not. However, we observed that the ATP regeneration system
tended to reduce the magnitude of the delay. In contrast to
H+ pumping, ATP hydrolysis did not show a similar lag phase
(data not shown). This suggests that in the presence of
KNO3, the fruit proton pumps were initially uncoupled, but
became progressively coupled as catalysis progressed.
Sensitivity to Vanadate--
The lemon fruit V-ATPase was
previously found to be partially sensitive to high concentrations
(
As shown in Fig. 4, A and C, respectively, proton
pumping and ATP hydrolysis by tonoplast vesicles of epicotyls exhibited little sensitivity to vanadate in the 0-50 µM range.
Thus, the deduced coupling ratio of the epicotyl V-ATPase increased
only slightly between 10 and 50 µM vanadate. The lack of
effect of vanadate suggests that contaminating P-ATPase activity is not a significant factor in our measurements of the coupling ratio of the
epicotyl V-ATPase (Fig. 4E). High concentrations of vanadate inhibited proton pumping to some extent, while ATP hydrolysis appeared
to be stimulated under the same conditions. However, this latter effect
appeared to be an artifact due to the oxidative properties of vanadate
which, at high concentration, oxidized the NADH used in the coupled
assay (data not shown).
In contrast to the results with tonoplast vesicles from epicotyls, ATP
hydrolysis by lemon fruit membranes was inhibited by ~50% by 50 µM vanadate (Fig. 4D). However, proton pumping
was only slightly inhibited by low concentrations of vanadate in both the nitrate-sensitive and the nitrate-insensitive preparations. This
suggests that a considerable amount of contaminating plasma membrane
ATPase activity, presumably in the form of leaky vesicles incapable of
generating a V1 Dissociation--
We previously reported that cold
dissociation in the presence of KNO3 caused the release of
catalytic subunits from fruit vesicles without affecting the total
ATP-dependent H+ pumping activity relative to
controls (13). The only effect of nitrate treatment on the fruit
H+ pumping activity was to increase the sensitivity to high
concentrations of vanadate (200 µM) from 30% inhibition
in control vesicles (treated with cold an 5 mM MgATP alone)
to 60-70% in vesicles treated with cold 5 mM MgATP
and 500 mM KNO3. Fig.
5 confirms that the amount of catalytic
subunit released from fruit membranes by nitrate was comparable to the
amount dissociated from epicotyl vesicles, as determined by
immunoblotting. The ATP-dependent proton pumping activity,
measured in the absence of vanadate, is given below the
immunoblots.
To determine whether the H+/ATP coupling ratio of the fruit
V-ATPase had been altered by the treatment with 500 mM
KNO3, nitrate-insensitive and nitrate-sensitive fruit
vesicle preparations were subjected to cold inactivation in the
presence or absence of 500 mM KNO3. H+ pumping and ATP hydrolysis were measured simultaneously
after washing the cold-released vesicles with buffer. The results are shown in Fig. 6 (A to
D). For a comparison, the activity of similarly treated
epicotyl vesicles is also presented (Fig. 6, E and
F). In the nitrate-insensitive fruit preparation,
KNO3 treatment had little or no effect on either the
H+ pumping or ATP hydrolysis activities (Fig.
6A) and thus, the coupling ratio of the nitrate-insensitive
preparation was unaffected. However, the nitrate-treated sample was
strongly inhibited by 500 µM vanadate (Fig.
6B). The results obtained with the nitrate-sensitive fruit
preparation confirmed these findings (Fig. 6, C and
D) and showed that 50 µM vanadate was
sufficient to inhibit most of the proton pumping activity after
KNO3 treatment. Table II
shows a comparison of the H+ pumping and hydrolytic
activities, and the coupling ratios of the "partially
nitrate-sensitive" fruit preparation used in Fig. 6, C and
D. Under control conditions (non-dissociated), the
vanadate-sensitive ATPase activity was largely independent of
H+ pumping, since 50 µM vanadate inhibited
ATP hydrolysis by 58% and H+ pumping by only 16%.
However, after V1 dissociation, 50 µM
vanadate inhibited ATP hydrolysis and H+ pumping by 71 and
74%, respectively, indicating that the vanadate-sensitive ATP
hydrolytic activity is coupled to proton transport. Since the amount of
vanadate-sensitive H+ pumping activity was doubled in
absolute value after V1 dissociation (0.27 to
0.54 arbitrary units), the dissociation procedure appears to have
partially transformed the vanadate-insensitive H+ pumping
activity into a vanadate-sensitive activity. Overall, the
H+/ATP coupling ratio of the partially nitrate-sensitive
preparation, which in the control was 2.66 a.u. in the presence of
50 µM vanadate, dropped to 0.61 ± 0.04 a.u.
after V1 dissociation, whether vanadate was present or not
(Table II). Thus V1 dissociation has a major uncoupling
effect on the nitrate-sensitive H+ pumping activity of
juice sac vesicles.
A detailed analysis of vanadate sensitivity and coupling ratios was
also performed under control and V1 dissociation conditions for a nitrate-sensitive fruit preparation (Fig.
7). In the control vesicles, 50 µM vanadate inhibited proton pumping by only 27% (Fig.
7A), whereas ATP hydrolysis was inhibited by 53% by the same concentration (Fig. 7B). After V1
dissociation, 50 and 100 µM vanadate inhibited the proton
pumping activity by 69 and 89%, respectively (Fig. 7A). The
H+/ATP coupling ratio, which in the control was ~1.8 a.u.
in the presence of 50 µM vanadate, dropped to ~0.62
a.u. after V1 dissociation (Fig. 7C). At 400 µM vanadate the coupling ratio of the control was ~2.3,
while that of the V1 dissociated membranes was ~0.1. Thus, high concentrations of vanadate have opposite effects on the
coupling ratios of control versus V1 dissociated
vesicles, increasing the former while decreasing the latter.
ATP Kinetics--
Since the residual H+-ATPase
activity after V1 dissociation treatment clearly differed
from the control activity in terms of vanadate sensitivity, both
activities were further characterized with respect to
Km and Vmax in a fruit
preparation exhibiting ~40% sensitivity to 100 mM
KNO3 (Fig. 8). From the
curves in Fig. 8B, it is clear that the ATP hydrolytic
activity was largely unaffected by nitrate up to a substrate
concentration of 2 mM ATP. Beyond 2 mM, the
first-order kinetics of the control vesicles diverged from strict
Michaelis-Menten kinetics, suggesting the presence of some
contaminating activity (Fig. 8B). From the Hanes-Woolf linearizations of the first-order kinetics between 0 and 2 mM ATP, we calculated an identical Km of
0.16 mM ATP and an identical Vmax
for the vesicles treated in the presence or absence of KNO3
(Fig. 8D). In contrast to ATP hydrolysis, the Vmax of the proton pumping activity was reduced
50% by nitrate treatment (Fig. 8, A and C).
However, the Km of 0.20 mM ATP for the
nitrate-treated sample was identical to that of the control, suggesting
that the same type of enzyme was active after V1
dissociation as before. In order to determine whether the treatment
with nitrate might have left intact a population of V-ATPases with a
different coupling ratio than the normal V-ATPase, we normalized the
initial rates of pumping of control and nitrate-treated vesicles and
compared their pH gradient at steady state (Fig. 9A). The Uncoupling by Nitrate--
Experiments were carried out to
determine whether the epicotyl V-ATPase and the nitrate-sensitive
component of the fruit V-ATPase exhibited the same pattern of
uncoupling by nitrate. Because acridine orange was reported to
dissipate pH gradients in the presence of KNO3 (20), we
used quinacrine fluorescence quenching to measure proton transport in
the experiments where nitrate was present.
As shown in Fig. 10, A and
B, nitrate inhibited the H+ pumping and ATP
hydrolysis activities of both the epicotyl and the fruit tonoplast
vesicle preparations, although the total inhibition was greater for
epicotyl vesicles. The average coupling ratios of two fruit and two
epicotyl preparations in the presence of increasing nitrate
concentrations are shown in Fig. 10C. Note that only the
nitrate sensitive activity was considered. The progressive decrease in
the H+/ATP coupling ratio between 0 and 50 mM
KNO3 clearly indicates that in fruit and epicotyl vesicles,
nitrate inhibits proton pumping to a greater extent than ATP
hydrolysis, and thus uncouples the V-ATPase. If only the
nitrate-sensitive portion of the total activity is considered,
uncoupling of the fruit V-ATPase occurs more readily at very low
concentrations of KNO3 (<10 mM); at
concentrations >20 mM, however, the epicotyl is more
strongly uncoupled by nitrate than the fruit. In the presence of
50-100 mM KNO3, the H+/ATP
coupling ratio of the fruit tonoplast vesicles was consistently about
twice that of the epicotyl vesicles.
Bafilomycin A1--
Similar experiments were carried
out to measure the effect of bafilomycin A1 on the coupling
ratios of fruit and epicotyl tonoplast membrane vesicles (Fig.
11, A and B). In
preliminary experiments it was found that fruit preparations that were
more nitrate-sensitive exhibited increased sensitivity to bafilomycin as well. Fig. 11, A and B, show the effects of
bafilomycin on the H+ pumping and ATP hydrolysis activities
of an epicotyl preparation and a bafilomycin-sensitive fruit tonoplast
preparation, respectively. The H+/ATP coupling ratios
between 0 and 1 µM bafilomycin are shown in Fig.
11C. Bafilomycin had no effect on the coupling ratios of either the epicotyl or the fruit V-ATPases.
Oxidation--
We previously demonstrated that oxidation inhibits
the proton pumping activity of the epicotyl V-ATPase, and that the
oxidative inactivation is partially reversible by DTT (13).
Accordingly, the effect of oxidation on the H+/ATP coupling
ratios of epicotyl tonoplast vesicles was examined. To avoid artifacts
due to contaminating ATPases, only the bafilomycin- and NEM-sensitive
ATP hydrolysis and proton pumping activities were used for the
determination. Exposing epicotyl tonoplast vesicles to air at 22 °C
in the absence of reductant reduced both H+ pumping and ATP
hydrolysis activities in parallel (Fig.
12A). Moreover, DTT reversal
was the same in both cases. The calculated coupling ratios are shown in
Fig. 12B. Oxidation had no effect on the H+/ATP
coupling ratios of epicotyl V-ATPases.
V-ATPases normally operate far from thermodynamic equilibrium and
are therefore considered to be under kinetic regulation (e.g. 11). Kinetic regulation of the V-ATPase may involve
inhibitors (21, 22), V1 dissociation from the membrane
(23), slip induced by We previously reported that ATP-dependent proton pumping by
juice sac vesicles was unusually insensitive to nitrate and other V-ATPase inhibitors (13). Although the juice sac V-ATPase became sensitive to inhibitors after being solubilized, purified, and reconstituted into liposomes, its pH-dependent slip rate
was still about one-half that of the epicotyl V-ATPase. We therefore
proposed that the low slip rate of the juice sac V-ATPase might be one of the factors which allows the lemon fruit V-ATPase to build up its
steep equilibrium pH gradient (14).
In this report we have shown that the nitrate insensitivity of proton
pumping by the lemon fruit V-ATPase is not constant throughout the
year, but exhibits a seasonal variation. The minimum nitrate
sensitivity occurs during the winter months, and the maximum nitrate
sensitivity occurs in the spring and early summer. Since the nitrate
sensitivity of the ATP hydrolytic activity is relatively constant
throughout the year, what varies is the coupling between H+
transport and ATP hydrolysis. Whether this variation is due to changes
in rainfall or other environmental factors remains to be determined.
Since we have shown that nitrate insensitivity was
membrane-dependent, it is possible that a seasonal change in membrane lipids is driving the seasonal fluctuation in nitrate sensitivity. Accordingly, the variable nitrate sensitivity may be
associated with a single proton pump, the V-ATPase, which may exist in
two states: a nitrate-sensitive (normal) state and a nitrate-insensitive (altered) state. These two states make up two
subpopulations of V-ATPases on the juice sac tonoplast, with the
relative amounts of each varying with the seasons. We designate this
situation as the "one pump/two states model" in contrast to a
"two pumps" model which involves the presence of two distinct H+-ATPases with different stoichiometries.
One possible way to distinguish between proton pumps with different
H+/ATP stoichiometries is to compare their
H+/ATP coupling ratios, assuming tight coupling between
hydrolysis and proton transport. In the present study, we used initial
activity rates to compare the H+/ATP coupling ratios of the
proton pumps in fruit and epicotyl tonoplast vesicles.
A comparison of nitrate-sensitive versus nitrate-insensitive
fruit preparations indicates that nitrate-sensitive vesicles have
higher H+/ATP coupling ratios than nitrate-insensitive
preparations. Since the coupling ratio of the nitrate-sensitive
vesicles is comparable to that of epicotyl preparations, and assuming
that the epicotyl tonoplast is energized by a V-ATPase alone, the
nitrate-sensitive juice sac vesicles are energized by only one type of
proton pumping ATPase: the V-ATPase. If it contained a mixture of P-
and V-type ATPases, with H+/ATP stoichiometries of 1 and 2, respectively (11, 25-27), one would expect the coupling ratio of the
fruit preparation to be lower than that of the epicotyl.
Even though the coupling ratio of the so-called nitrate-insensitive
juice sac preparations was lower than that of tonoplast vesicles from
epicotyls, two observations suggest that these vesicles also bear a
V-ATPase rather than another type of H+-ATPase: 1) during
the initial seconds of H+ pumping in the presence of
nitrate, the nitrate-insensitive fruit preparations are temporarily
uncoupled. The subsequent recovery of coupling is dependent on the
presence of MgATP and may involve some type of subunit rearrangement or
possibly a phosphorylation reaction.2 2)
Nitrate-insensitive juice sac preparations show kinetic properties and
vanadate sensitivities similar to the more nitrate-sensitive juice sac
preparations after V1 dissociation treatment. In the latter
case, as shown below, the residual H+ pumping activity is
thought to be due to a V-ATPase, based on its Km and
the simultaneously measured ATP hydrolysis kinetics, even though the
calculated coupling ratio is lower than in epicotyl V-ATPases.
Moriyama and Nelson (10) have proposed that nitrate induces uncoupling
of proton transport from ATP hydrolysis in V-ATPases. In
tonoplast-enriched vesicles from lemon juice sacs and epicotyls, nitrate indeed had such an effect. When only the nitrate-sensitive components of H+ pumping and ATP hydrolysis were considered
(defined as the portions of the activities inhibited by 400 mM KNO3), juice sac and epicotyl preparations
exhibited significant uncoupling under treatment with 1 to 100 mM KNO3. At 50 mM KNO3,
the H+/ATP coupling ratio of epicotyl V-ATPases had dropped
an average 89% from its initial value, while the fruit enzymes'
uncoupling averaged ~72% of their initial coupling ratio.
Accordingly, a subpopulation of nitrate-sensitive V-ATPases requires
400 mM KNO3 for inhibition while remaining
resistant to uncoupling by 50 to 100 mM
KNO3.
Although the H+ pumping activity of fruit vesicles becomes
more sensitive to vanadate after nitrate treatment, in untreated vesicles vanadate is most effective in the 50 to 300 µM
range. Since P-type H+-ATPases are generally inhibited by
1.0 to 10 µM vanadate, it is unlikely that the vanadate
sensitivity of the lemon fruit H+ pumping activity involves
the inhibition of a P-type enzyme (14). However, an ATP hydrolytic
activity that is sensitive to low concentrations of vanadate is clearly
present in fruit vesicle preparations. This is best illustrated by the
H+/ATP coupling ratios in the presence of increasing
concentrations of vanadate. For nitrate-sensitive and
nitrate-insensitive juice sac preparations, the coupling ratios rise
sharply in the presence of 0 to 50 µM vanadate, and then
remain stable up to 500 µM VO4. This
indicates that the "contaminating" activity is mainly hydrolytic, and as it can be eliminated by low concentrations of vanadate (<50
µM), it could be due to a P-type ATPase. Since
concentrations of vanadate between 50 and 500 µM
inhibited the hydrolytic and pumping activities to the same extent,
i.e. the H+/ATP coupling ratio remains stable,
high concentrations of vanadate appear to inhibit a single
H+-ATPase activity which is distinct from the activity
inhibited by low concentrations of vanadate.
V1 dissociation treatments in the presence of high nitrate
did not significantly modify the H+/ATP coupling ratio in
nitrate-insensitive juice sac preparations. However, V1
dissociation did increase the sensitivity of the proton pump to high
concentrations of vanadate. In nitrate-sensitive preparations,
H+/ATP coupling ratios were strongly reduced by the
V1 dissociation treatment, and concomitantly, proton
pumping became highly sensitive to both high and low
concentrations of vanadate. In the preparations analyzed, which were
initially ~20% sensitive to vanadate, KNO3 treatment
increased the vanadate sensitivity to 50 to 70% of the initial
activity. Simultaneously, the coupling ratio dropped to 1/2 or 1/4 of
its value before KNO3 treatment. Three explanations are
possible. 1) KNO3 treatment uncouples the V-ATPases present on the membranes while simultaneously coupling a population of P-type
H+-ATPases which were initially uncoupled. 2)
KNO3 treatment uncouples one of two populations of
V-ATPases, the remaining population being vanadate-sensitive. 3)
KNO3 uncouples one of two populations of V-ATPases and
modifies the remaining V-ATPases, inducing them to become
vanadate-sensitive.
While the first explanation would yield a result similar to what was
observed, it would imply that nitrate treatment induces the sealing of
a leaky membrane fraction associated with the putative P-type
hydrolytic activity. To our knowledge, no such effect of nitrate on the
formation of sealed vesicles has been reported. Thus, we consider this
explanation unlikely.
In the second explanation, there are two populations of V-ATPases:
classical, nitrate-sensitive V-ATPases and nitrate-insensitive, vanadate-sensitive V-ATPases. However, this explanation does not account for the overall increased vanadate sensitivity of proton pumping after V1 dissociation treatment.
The third explanation differs from the second in that a population of
V-ATPases becomes vanadate-sensitive as a result of nitrate treatment.
The increase in vanadate sensitivity could be a consequence of a
molecular rearrangement that allows the fruit V-ATPase to continue to
pump protons in the presence of nitrate. This explanation is consistent
with previous results, in which we found that a vanadate-sensitive
ATPase activity, apparently associated with partially disassembled
V-ATPases, co-migrated with the juice sac V-ATPase during its
purification by gel filtration and anion exchange chromatography (14).
Thus, according to this third explanation, after some fruit V-ATPases
become damaged by KNO3 treatment, their activity is
modified so as to become more vanadate-sensitive, while the overall
coupling ratio is reduced due to uncoupling of another subpopulation of
V-ATPases. The same type of damage may occur in vivo during
certain periods of the year, resulting in nitrate-insensitive juice sac
proton pumps and a low H+/ATP coupling ratio. This one
pump/two states model is further supported by the finding that the
Km values for MgATP were identical in control
vesicles and in vesicles treated for V1 dissociation. This
strongly suggests that the same type of H+-ATPase was
active before and after treatment with nitrate. The fact that the
measured Km values were in the 0.16 to 0.20 mM range, considerably lower than the 0.88 ± 0.18 mM previously determined for the juice sac H+
pumping activity (13) is attributed to the presence of an ATP regenerating system in the present study, which converts even traces of
ADP back into substrate, while removing a potent inhibitor of the
V-ATPase. Moreover, since the equilibrium It has been argued that inhibition of the V-ATPase by nitrate was the
result of two distinct phenomena (28, 29). At high concentrations, the
chaotropic properties of nitrate promote the dissociation of the
V1 sector from Vo (28), while below 50 mM, nitrate was proposed to involve a different mode of
action (29, 30). Our present results indicate that there is no distinct low- and high-nitrate effects on the lemon V-ATPases, as from 0 to 400 mM KNO3, the effect of nitrate corresponds to
the progressive uncoupling of V1 from Vo which
ends with the physical dissociation of the V1 sector from
the membrane. Note that even after treatment of epicotyl vesicles with
500 mM KNO3, ~50% of the V1
sectors remain attached to the membrane, even though proton pumping is completely inhibited. Thus, physical release of the V1
sector from the membrane is not required for inhibition of proton
pumping, but may be required for inhibition of ATP hydrolysis.
Dschida and Bowman (31) proposed that the low concentration effect of
nitrate was due to its oxidizing properties and induced the formation
of disulfide cross-links in V1 subunits, conformational changes, and subsequent release of V1 sectors from the
membrane. Accordingly, the oxidative inactivation of proton pumping
that we had observed in epicotyl vesicles may not have been due
exclusively to disulfide bonding at the catalytic site (13, 14, 32, 33), but could also have involved some oxidation-induced uncoupling of
the enzyme (34). Furthermore, sulfhydryl groups of the Bafilomycin A1 is thought to inhibit V-ATPases by binding
to the 100-kDa subunit and/or the proteolipid of the Vo
sector, but its mode of action, as well as that of other macrolide
antibiotics, is still unknown (36-38). We found that, like oxidation,
bafilomycin A1 did not uncouple H+ pumping from
ATP hydrolysis.
It is important to note that in the present study H+/ATP
coupling was measured in the absence of an electrical potential
gradient and in the presence of a limited pH gradient developing during the initial few minutes of H+ pumping. H+/ATP
coupling ratios under these initial rate conditions might differ
drastically from those encountered under the conditions prevailing
in vivo, e.g.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pH than the pump with the lower coupling ratio. Thus the
coupling ratio of the lemon fruit V-ATPase might be higher than that of the epicotyl V-ATPase even if the two pumps had the same stoichiometry. To test this hypothesis, we have carried out experiments to
characterize the coupling ratios of the fruit and epicotyl V-ATPases
under initial rate conditions. Moreover, since the H+
pumping activity of the fruit is relatively insensitive to
V1 dissociation, we also determined the effect of
V1 dissociation treatment on the coupling ratio of the
juice sac proton pump(s). Our results show that, in contrast to
pH-dependent slip, the H+/ATP coupling ratios
of the juice sac and epicotyl proton pumps, determined under initial
rate conditions, are the same. Whereas V1 dissociation with
nitrate induced the complete uncoupling of the epicotyl V-ATPase, only
a fraction of the fruit H+ pumping activity was uncoupled
under identical conditions. However, the kinetic properties of the
juice sac proton pumps after V1 dissociation treatment were
identical to those in the control vesicles. This strongly suggests that
in juice sac vesicles, the residual proton pumping activity after
V1 dissociation treatment belongs to a nitrate-resistant
subpopulation of V-ATPases, rather than to a different type of proton pump.
pH across the tonoplast of
juice sacs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Simultaneous measurement of ATP hydrolysis
and H+ pumping by tonoplast-enriched vesicles from juice
sacs. ATP hydrolysis was measured by coupling the oxidation of
NADH, measured at 340 nm (- - - -), to the appearance of ADP; and
proton pumping was measured as the absorbance quenching of acridine
orange at 495 nm ( ). Once a steady state
pH was reached, the
proton gradient was collapsed with 4 µM gramicidin or
0.25 µM nigericin. 20-25 µg of juice sac or
epicotyl membrane protein were typically used in all experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sensitivity of the Citrus limon V-ATPases to nitrate and
bafilomycin A1
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Fig. 2.
Inhibition of the proton pumping activity of
tonoplast-enriched juice sac vesicles by nitrate. The effect of
increasing concentrations of potassium nitrate on the maximum rate of
proton pumping by three different preparations of tonoplast-enriched
membranes from juice sacs was measured by quinacrine fluorescence
quenching. ~80 µg of membrane protein were used in each reaction.
represents a so called nitrate-insensitive preparation;
represents a nitrate-sensitive preparation;
a partially
nitrate-sensitive preparation.
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Fig. 3.
Seasonal variation of the nitrate sensitivity
of tonoplast-enriched juice sac vesicles. A, quinacrine
fluorescence quenching was used to measure the sensitivity of
tonoplast-enriched fruit vesicle preparations to 100 mM
potassium nitrate over a period of 20 months. All fruits were harvested
at the same stage of development from the same tree. For each membrane
preparation the H+ pumping activity was measured in the
presence and absence of 100 mM KNO3 and the
activity of the nitrate-treated samples, expressed as a percentage of
the control samples, is reported. B, initial rates of
quinacrine fluorescence quenching curves of a juice sac preparation
showing little sensitivity to nitrate. C, control;
+100 mM NO3, treated with potassium
nitrate.
200 µM) of vanadate (13, 14). We therefore tested the
effects of vanadate on the initial rates of proton pumping (Fig.
4, A and B) and ATP
hydrolysis (Fig. 4, C and D), and on the deduced
coupling ratio (Fig. 4, E and F) of tonoplast
vesicles from juice sacs and epicotyls. Curves representing two types
of juice sac preparations are shown, a "nitrate sensitive" one
(open circles), which was ~70% inhibited by 100 mM KNO3, and a "nitrate insensitive"
preparation (open triangles), which was inhibited only
~30% by the same concentration of nitrate.
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Fig. 4.
Effect of vanadate on H+
pumping, ATP hydrolysis, and the H+/ATP coupling ratios of
tonoplast-enriched vesicles from epicotyls and juice sacs.
H+ pumping (A and B) and ATP
hydrolysis (C and D) were measured
simultaneously, and the H+/ATP coupling ratios determined
(E and F) in epicotyl vesicles (A,
C, and E) and in juice sac vesicles
(B, D, and F). Two typical juice sac
preparations were used, a KNO3-sensitive one which was
inhibited ~70% by 100 mM KNO3 ( ) and a
KNO3-insensitive one which was inhibited ~30% by 100 mM KNO3 (
).
pH, may be present in the fruit tonoplast-enriched preparations. Both ATP hydrolysis and proton pumping were inhibited by
400 µM vanadate to ~60% of their activity in the
presence of 50 µM vanadate. The nitrate-sensitive fruit
preparation was about twice as active in proton pumping as the
nitrate-insensitive activity. As a result, the coupling ratio
calculated for the nitrate-sensitive preparation is about 70% higher
than that of the nitrate-insensitive preparation, and is comparable to
the coupling ratio of the epicotyl V-ATPase (Fig. 4F).
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Fig. 5.
V1 dissociation in
tonoplast-enriched vesicles of juice sacs and epicotyls. Epicotyl
and juice sac vesicles were treated with 0 or 500 mM
KNO3, under conditions which promote the dissociation of
the V1 complexes from the membrane. Following
V1 dissociation treatment, the vesicles were centrifuged
and equal volume fractions of the pellets and supernatants were
separated by SDS-PAGE, transferred to nitrocellulose, and probed with
antibodies to the catalytic subunit of the corn V-ATPase. Underneath
the immunoblots showing the amount of cross-reacting antibody are given
the residual H+ pumping activities in the membrane pellets,
expressed as a percentage of the activities of the membranes treated in
the absence of nitrate.
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Fig. 6.
Proton pumping and ATP hydrolysis by
tonoplast-enriched vesicles after V1 dissociation
treatment. A nitrate-insensitive juice sac preparation, a
partially nitrate-sensitive juice sac preparation, and an epicotyl
preparation were used for V1 dissociation treatment in the
presence or absence of 500 mM KNO3. Proton
pumping (curves) and ATP hydrolysis (lines) are
reported in A, C, and E. For the sake
of clarity, only the H+ pumping curves are shown in
B, D, and F. A and B
represent a nitrate-insensitive juice sac preparation; C and
D, a partially nitrate-sensitive juice sac preparation;
E and F, an epicotyl preparation. - - - -,
C. A, C, and E are the
controls treated in the absence of nitrate; , V1R,
A, C, and E represent the
KNO3-treated samples; - - - -, C + 50 µM
VO4, D and F are controls containing
50 µM vanadate in the reaction mixture; - - - -, C + 500 µM VO4, B, D, and
F are controls containing 500 µM vanadate in
the reaction mixture; - · - · - ·, V1R + 50 µM VO4, represent KNO3-treated
samples containing 50 µM vanadate in the reaction
mixture; ---, V1R + 500 µM VO4:
B, D, and F represent
KNO3-treated samples containing 500 µM
vanadate in the reaction mixture.
Activities and coupling ratios of fruit tonoplast vesicles treated for
V1 dissociation
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Fig. 7.
Effect of V1 dissociation
treatment on the vanadate sensitivity of a partially nitrate-sensitive
juice sac preparation. Tonoplast-enriched vesicles were treated
for V1 dissociation in the presence or absence of 500 mM KNO3. After washing in buffer, the
membrane-bound enzymes were assayed for H+ pumping and ATP
hydrolysis. Initial activity rates and coupling ratios in the presence
of increasing concentrations of vanadate are reported. A,
H+ pumping activities; B, ATPase activities; and
C, H+/ATP coupling ratios. , vesicles treated
in the absence of nitrate;
, vesicles treated in the presence of 500 mM KNO3.
pH built up by
nitrate-treated vesicles did not appear to differ significantly from
that of control vesicles. The pH-dependent slip and leakage
rate constants of control and nitrate-treated vesicles were calculated
and normalized for the apparent proton pumping rate at any time during
the
pH build up. As shown in Fig. 9B, the
pH-dependent slip and leakage were identical in control and
nitrate-treated vesicles.
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Fig. 8.
Kinetic analysis of H+ transport
and ATP hydrolysis in fruit tonoplast vesicles pretreated for
V1 dissociation in the presence or absence of nitrate.
Tonoplast-enriched juice sac vesicles were treated for V1
dissociation in the presence or absence of 500 mM
KNO3. After washing in buffer, 30 µg of membrane-bound
enzymes were assayed in the absence of vanadate for simultaneous proton
pumping and ATP hydrolysis in the presence of increasing concentrations
of MgATP. Initial rates of H+ pumping activities
(A) and ATP hydrolysis rates (B) are reported for
vesicles treated in the presence ( ) or absence (
) of nitrate.
Panels C and D represent the Hanes-Woolf
linearizations between 0 and 2 mM ATP of the first-order
kinetics in A and B, respectively.
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Fig. 9.
ATP-dependent proton pumping by
fruit vesicles pretreated for V1 dissociation in the
presence or absence of nitrate. A, tonoplast-enriched
juice sac vesicles, treated as described in the legend to Fig. 8, were
normalized for similar initial rates of H+ pumping. 7.5 µg of membrane protein for the control (- - - -) and 15 µg of
the nitrate-treated vesicles (---) were allowed to reach steady state
equilibrium pH (no vanadate was present in the reaction mixture).
Then the pH gradient was collapsed with 0.25 µM
nigericin. B, the pH-dependent slip and leakage
rate constants were determined as described previously (14) and
corrected for the rates of proton pumping by dividing them at any time
point by the derivative of a fifth order equation describing the
pumping curves obtained in panel A (= pumping rate).
- - - -, control; ---, nitrate-treated.
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Fig. 10.
Effect of nitrate treatment on the
H+ pumping and ATP hydrolysis activities, and on the
H+/ATP coupling ratios of epicotyl and juice sac
vesicles. Tonoplast-enriched vesicle preparations from epicotyls
and nitrate-sensitive fruit juice sacs were tested by quinacrine
fluorescence quenching for the nitrate sensitivity of their proton
pumping activity, and by a coupled assay for their ATPase activity.
Initial activity rates and coupling ratios are presented. A,
epicotyl vesicles; B, juice sac vesicles;
- - - - and
- -
- -, H+ pumping;
-
- and
-
-, ATP hydrolysis; C,
H+/ATP coupling ratios of the nitrate-sensitive components
of the pumping and hydrolytic activities (
, epicotyl;
, juice
sac).
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Fig. 11.
Effect of bafilomycin A1 on the
H+ pumping and ATP hydrolysis activities, and on the
H+ATP coupling ratios of epicotyl and juice sac
vesicles. Nitrate-sensitive tonoplast-enriched vesicle
preparations from epicotyls and juice sacs were tested for bafilomycin
sensitivity by simultaneously measuring proton pumping and ATP
hydrolysis. Initial activity rates and coupling ratios are presented.
A, epicotyl vesicles; B, juice sac vesicles;
- - - - and
- -
- -, H+ pumping;
-
- and
-
-, ATP hydrolysis; C,
H+/ATP coupling ratios of the bafilomycin
A1-sensitive components of the pumping and hydrolytic
activities (
, epicotyl;
, juice sac).
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Fig. 12.
Oxidative inactivation of the H+
pumping and ATPase activities of the epicotyl V-ATPase.
Tonoplast-enriched vesicles from epicotyls were incubated at 22 °C
in the absence of reductant, and their H+ pumping and ATP
hydrolysis activities were assayed at different time intervals in the
presence or absence of 50 nM bafilomycin A1 or
200 µM NEM. After ~4 h, 50 mM DTT were
added to an aliquot of the oxidized membranes. A, activity
measurements; , H+ pumping activity;
, bafilomycin
A1-sensitive ATPase activity;
, NEM-sensitive ATPase
activity; B, deduced coupling ratios;
, bafilomycin
A1-sensitive measurements;
, NEM-sensitive measurements.
Note: the H+ pumping activity was 100% sensitive to
bafilomycin A1 and NEM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
µH+ (10), and
variable H+/ATP coupling (24). In lemon juice sacs, the
steep trans-tonoplast pH gradient implies either that the V-ATPase
operates close to thermodynamic equilibrium, or that a second
H+-ATPase with a lower H+/ATP stoichiometry
than the V-ATPase is present on the membrane. For the V-ATPase to reach
thermodynamic equilibrium it would have to become refractory to kinetic regulation.
pH was unchanged by
treatment with nitrate, and since the calculated
pH-dependent slip and leakage rate was identical to that of
the control, KNO3 treatment did not reveal a V-ATPase with
a different stoichiometry. Based on all the above and consistent with
the one pump/two states model, we interpret the seasonal reduction in
the H+/ATP coupling ratio as representing a change in the
state of the V-ATPase, rather than a fluctuation in the activity of a
second type of proton pump on the tonoplast.
subunit of
the lettuce chloroplast F-ATPase have been shown to be involved in
proton slip (35). Our results, obtained by simultaneously measuring
bafilomycin- and NEM-sensitive H+ pumping and ATP
hydrolysis at different time points during oxidative inactivation show
that no H+/ATP uncoupling was taking place during oxidative
inactivation. Even though this does not preclude nitrate from having a
specific oxidizing effect, it indicates that oxidative inactivation, as we were measuring it previously, does not uncouple the enzyme and
probably takes place exclusively at the catalytic site.
= +20 mV;
pH = 2 to 4.5 pH units. According to Tu et al. (15),
and our own findings (14), intrinsic uncoupling, or slip, is
proportional to the pH gradient built up across the membrane. When slip
is determined during the attainment of a steady state pH gradient, the
fruit V-ATPase was shown to exhibit half as much slip as the epicotyl
V-ATPase (14). Thus, a lower rate of slip in the presence of a pH
gradient remains the most likely factor allowing the generation of
a steeper
pH by the fruit V-ATPase. Although the coupling ratios of
the fruit and epicotyl V-ATPases measured in the absence of a pH
gradient were equal, the fact that the V-ATPase of juice sacs retained some coupling in the presence of nitrate, whereas the epicotyl V-ATPase
did not, may be significant biologically. Nitrate may be mimicking the
effect of another stress, such as low cytosolic pH, which would tend to
inactivate a normal V-ATPase. The ability of the fruit V-ATPase to
adjust to adverse conditions may be a key factor in the overall
regulation of vacuolar pH in lemon.
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ACKNOWLEDGEMENTS |
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We thank Julia Staley for technical assistance in the preparation of membranes. We also thank Dr. Rafael Ratajczak for help in the initiation of this project.
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FOOTNOTES |
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* This work was supported in part by Grant DE-FG03-84ER13245 (to L. T.) from the U. S. Department of Energy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Swiss National Foundation for
Scientific Research during part of this work.
§ To whom correspondence and reprint requests should be addressed. Tel.: 831-459-2036; Fax: 831-459-3139; E-mail: taiz{at}biology.ucsc.edu.
2 M. Jensen, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
V-ATPase, vacuolar
H+-ATPase;
a.u., arbitrary units;
BTP, 1,3-bis-(tris(hydroxymethyl)methylamino)pro-pane;
DTT, dithiothreitol;
Mes, 2-(N-morpholino)ethanesulfonic acid;
MOPS, 3-(N-morpholino)propanesulfonic acid;
NEM, N-ethylmaleimide;
PAGE, polyacrylamide gel electrophoresis;
RB, resuspension buffer;
µH+, proton
electrochemical gradient;
, membrane potential.
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