V-ATPase of Thermus thermophilus Is Inactivated
during ATP Hydrolysis but Can Synthesize ATP*
Ken
Yokoyama
,
Eiro
Muneyuki§,
Toyoki
Amano§,
Seiji
Mizutani,
Masasuke
Yoshida§,
Masami
Ishida¶, and
Shouji
Ohkuma
From the Department of Biochemistry, Faculty of Pharmaceutical
Science, Kanazawa University, Takara-machi 13-1, Kanazawa,
Ishikawa 920, the § Research Laboratory of Resources
Utilization, Tokyo Institute of Technology, Nagatsuta 4259,
Yokohama 226, and the ¶ Laboratory of Biochemistry of Marine
Resources, Tokyo University of Fishers, Konan 4, Minato-ku,
Tokyo 108, Japan
 |
ABSTRACT |
The ATP hydrolysis of the
V1-ATPase of Thermus thermophilus have
been investigated with an ATP-regenerating system at 25 °C. The
ratio of ATPase activity to ATP concentration ranged from 40 to 4000 µM; from this, an apparent Km of
240 ± 24 µM and a Vmax of
5.2 ± 0.5 units/mg were deduced. An apparent negative
cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase.
Interestingly, the rate of hydrolysis decayed rapidly during ATP
hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the
inactivation of the V1-ATPase was attained by a prior
incubation with ADP-Mg. The inactivated V1-ATPase contained
1.5 mol of ADP/mol of enzyme.
Difference absorption spectra generated from addition of ATP-Mg to the
isolated subunits revealed that the A subunit can bind ATP-Mg, whereas
the B subunit cannot. The inability to bind ATP-Mg is consistent with
the absence of Walker motifs in the B subunit.
These results indicate that the inactivation of the
V1-ATPase during ATP hydrolysis is caused by entrapping
inhibitory ADP-Mg in a catalytic site.
Light-driven ATP synthesis by
bacteriorhodopsin-VoV1-ATPase proteoliposomes
was observed, and the rate of ATP synthesis was approximately constant.
ATP synthesis occurred in the presence of an ADP-Mg of which
concentration was high enough to induce complete inactivation of ATP
hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction.
 |
INTRODUCTION |
VoV1-ATPases and
FoF1-ATPases constitute two subclasses of the
ATPase/ATP synthase superfamily (1, 2).
VoV1-ATPases are present in the membranes of
lysosomes (3), clathrin-coated vesicles (4), chromaffine granules (5),
and the central vacuoles of yeast (6). They are responsible for
vacuolar acidification, which plays an important role in a number of
cellular processes (1). VoV1-ATPases are also
found in the plasma membranes of most archea (7-9) and some kinds of
eubacteria (10-12). Several studies indicate that the physiological
role of VoV1-ATPases of some archea and the
thermophilic eubacterium Thermus thermophilus is ATP
synthesis coupled to proton flux across the plasma membranes (7, 9,
13-15).
VoV1-ATPases consist of two functional
assemblies, a peripheral V1 moiety and a membrane
integrated Vo moiety, which are counterparts of the
F1 and Fo moiety of the
FoF1-ATPase (1, 15-17). The peripheral V1 moiety is composed of two major subunits, A and B, and
other minor subunits. Both structural analysis and sequence homology indicate an evolutionary relationship between
VoV1-ATPases and FoF1-ATPase and that the A and B subunit of
VoV1-ATPase are homologous to the
and
subunit of FoF1-ATPases (2). The A subunit of VoV1-ATPases contains the Walker motifs (1),
which are critical for nucleotide binding (18, 19). Labeling of the A
subunit by 2-azido-[32P]ATP correlates well with
inactivation of ATPase activity, with complete inactivation observed
upon modification of a single A subunit per complex (20). These
findings indicate that the catalytic site of the
VoV1-ATPase is located on the A subunit. On the
other hand, the B subunit of VoV1-ATPases lacks
Walker motifs. A recent study reported that the B subunit in the
VoV1-ATPase of clathrin-coated vesicles was
modified by 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (21). However, any direct evidence for the nucleotide binding to the
isolated B subunit of the VoV1-ATPase has not
been reported yet.
Structural similarity and sequence homology of the major subunits of
VoV1-ATPases and
FoF1-ATPases lead to the hypothesis that the
mechanisms of ATP hydrolysis and ATP synthesis by
VoV1-ATPases are almost identical to those of
FoF1-ATPases. Nevertheless, the enzymatic
properties of VoV1-ATPases and
FoF1-ATPases are different (1). Whereas azide
inhibits ATP hydrolysis by F1-ATPases by stabilizing the
inhibitory ADP-Mg-F1-ATPase complex (22-24), it does not
inhibit ATPase activity of VoV1-ATPases (1,
10).
Precise understanding of VoV1-ATPases would
allow the comparison to FoF1-ATPases and the
elucidation of the common essential mechanism for the coupling of
proton translocation across a membrane with ATP formation. However,
several problems, such as the difficulty of obtaining a large amount of
pure enzyme from vacuolar membranes and an unstable V1
moiety (17), have limited our investigation of enzymatic properties of
VoV1-ATPases.
T. thermophilus, originally isolated from a hot spring in
Japan, is thermophilic, obligatory aerobic, Gram-negative, and
chemoheterotrophic eubacterium (25). Its respiratory chain may include
energy coupling Site I (26). This bacterium has a large amount of the
VoV1-ATPase on the plasma membrane, instead of
FoF1-ATPase (15).
In contrast to eukaryotic equivalents, the V1 moiety of
T. thermophilus is easily detached from the membranes using
chloroform treatment and ATPase-active stable complex can be obtained
in large amounts (10). Throughout this manuscript, the V1
moiety from T. thermophilus is refereed to
V1-ATPase.
The V1-ATPase consists of four kinds of subunit with
apparent molecular sizes of 66 (A or
), 55 (B or
), 30 (
), and
11 (
) kDa, which are present in a stoichiometry of
A3B3
1
1. Similar to its eukaryotic counterparts, the V1-ATPase also shows
enzymatic properties different from those of F1-ATPases,
such as low specific activity, high Km values, and
resistance to azide inhibition (10). We previously reported a specific
activity of the V1-ATPase of about 0.1 units/mg of protein
at 55 °C in the absence of an ATP-regenerating system.
In this report, we demonstrate the particular kinetic behaviors of the
V1-ATPase of T. thermophilus in the presence of
ATP regenerating system, the nucleotide binding properties of the isolated A and B subunits, and ATP synthesis of a
VoV1-ATPase co-reconstituted with
bacteriorhodopsin for the first time.
 |
EXPERIMENTAL PROCEDURES |
Materials--
V1-ATPase and
VoV1-ATPase were prepared from T. thermophilus plasma membranes using the previously described
methods (10, 15). Bacteriorhodopsin
(bR)1 was prepared from
Halobacterium halobium (27). [
-32P]ATP was
purchased from NEN Life Science Products. Radioactive ATP (9.25MeBq/ml)
was diluted with nonradioactive ATP to the desired specific activity.
Egg yolk phosphatidylcholine (type XVI-E) was purchased from Sigma.
Other chemicals were purchased from Nacarai Corp.
Construction of Overexpression Systems of the A and B Subunits
and Purification of the Products--
The DNA fragments of the A and B
subunits were PCR amplified using Ex TaqTM polymerase
(TaKaRa) to minimize errors. The sequence of the amplified DNA
fragments was confirmed using ABI 373A sequencer. The obtained DNA
fragments were ligated into pUC18 to result in vectors pATPA and pATPB,
respectively. After transformation in Escherichia coli
strain JM 103, the expression of subunit genes was induced by addition
of 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside.
More than 10% of the total soluble protein was the recombinant A
subunit after induction, about 5% in case of the recombinant B subunit
(see Fig. 5). Both subunits were purified by the same method. About
10 g of cells were obtained from an overnight culture, suspended
in 50 ml of buffer containing 50 mM Tris-SO4
(pH 8.0), 50 mM NaCl, and 0.1 mM EDTA, and
disrupted by sonication. The membrane fraction and cell debris were
removed by centrifugation, and the supernatant was applied onto a DEAE
Sephacel column (2 × 10 cm) equilibrated with Buffer A (50 mM Tris-Cl (pH 8.0) and 0.1 mM EDTA). The
column was washed with 200 ml of Buffer A, and the proteins were eluted
with linear NaCl gradient (0-0.5 M). Fractions containing
the recombinant subunit were chosen after SDS-PAGE analysis (15% of
acrylamide gels). The fractions were combined, and solid ammonium
sulfate was added to a final concentration of 1.2 M and
stirred for 1 h. After precipitation by centrifugation, the
supernatant was applied onto a butyl-toyopearl column (Tosoh Corp.;
2 × 10 cm). Proteins were eluted with a reverse ammonium sulfate
gradient (1.2-0 M). Fractions containing the subunit were combined, and proteins were precipitated by ammonium sulfate. The
precipitate was dissolved with a minimum volume of Buffer A, and it was
applied onto a Sephacryl S-300 gel permeation column (1 × 90 cm)
equilibrated with Buffer A plus 50 mM
Na2SO4. The column was eluted with the same
buffer and fractions containing the subunits were chosen after SDS-PAGE
analysis (15% of acrylamide gels). The purified subunits were stored
at 4 °C until use.
Analytical Methods--
The protein concentrations of
V1-ATPase were determined by measurement of absorbance at
280 nm using a factor of 0.59 for the absorbance of 1 mg/ml protein.
The factor of 0.59 was determined by quantitative total amino acid
analysis and spectral data. Unless otherwise specified, ATPase activity
was measured at 25 °C with an enzyme-coupled ATP-regenerating
system. The reaction mixture contained 50 mM Tris-Cl (pH
8.0), 100 mM KCl, different concentrations of ATP-Mg, 2.5 mM phosphoenolpyruvate, 50 µg/ml pyruvate kinase, 50 µg/ml lactate dehydrogenase, and 0.2 mM NADH in a final
volume of 1.2 ml. Typically, the reaction was started by addition of the enzyme dissolved in 50 mM Tris-SO4 (pH
8.0), 50 mM Na2SO4, 0.2 mM EDTA to 1.2 ml of the assay mixture, and the rate of ATP hydrolysis was monitored as the rate of oxidation of NADH determined by
the absorbance decrease at 340 nm. The data were stored in a computer
for further analyses. The spectrometer was equipped with a small
stirrer for rapid mixing and with a device that enabled us to start the
reaction by injecting the enzyme solution without opening the lid. We
confirmed that the maximum dead time was less than 5 s after
starting the reaction. A phosphate-molybdate assay was used for the
measurement of the ATPase activity in the presence of ADP. The standard
reaction mixture was prepared in a final volume of 0.5 ml, containing
4.5 mM ATP-Mg, 2 mM MgCl2, 100 mM KCl, and 50 mM Tris-Cl (pH 8.0). The
reaction mixture was preincubated for 3 min at 25 °C before starting
the reaction by addition of enzyme. The reaction was terminated with
0.3 ml of 3% perchloric acid, and the amount of Pi was
assayed as described previously (28).
Enzyme-bound nucleotide was analyzed by anion exchange high performance
liquid chromatography. Bound nucleotides were released from the enzyme
by addition of 5 µl of 60% perchloric acid to 50 µl of the enzyme
solution. Thereafter, the mixture was incubated on ice for 10 min.
Then, 5 µl of 5 M K2CO3 solution
were added to the mixture and incubated on ice for 10 min. The
resulting pellet was removed by centrifugation at 4 °C. The
supernatant was applied to a Cosmopak-200 column equilibrated with 0.1 M sodium-phosphate buffer (pH 7.0). The column was eluted
isocratically with the same buffer at a flow rate of 0.8 ml/min. The
nucleotide was monitored at 258 nm. The peak area was determined by
automatic integration.
Interaction of ATP with the isolated subunit was assayed by UV
difference spectra at 25 °C with a Double Beam Spectrophotometer model U-3200 (Hitachi Corp.) using a pair of matched double cells, as
described in Ref. 29. The subunits were dissolved in 10 mM Tricine-NaOH buffer (pH 8.0) at a final concentration of 10 µM.
Preparation of Proteoliposomes and Light-induced ATP
Synthesis--
Proteoliposomes containing bR and
VoV1-ATPase were reconstituted according to the
procedure by Richard et al. (30). The reconstitution was
performed at 25 °C in 25 mM potassium phosphate buffer
(pH 7.3), 50 mM K2SO4, and 50 mM Na2SO4. Unilamellar liposomes were prepared by reverse phase evaporation using phosphatidylcholine and resuspended at a lipid concentration of 4 mg/ml. Triton X-100 was
added to a final concentration of 8 mg/ml. bR was solubilized from
purple membranes with 2 mg/ml Triton X-100. Then, 50 µl of solubilized bR solution (5 mg of protein/ml) and 10 µl of
VoV1-ATPase solution (3 mg of protein/ml) were
added to 850 µl of liposome solution.
n-Octyl-
-D-glucopyranoside was added to a
final concentration of 20 mM, and the mixture was incubated
for 5 min. Then, pyranine (excitation, 450 nm; emission, 510 nm) was
added to the mixture at a final concentration of 0.2 mM.
The detergent was removed by four successive additions of 80 mg/ml
washed Bio-beads SM-2 (Bio-Rad). The measurement of the fluorescence of
pyranine trapped inside the liposomes confirmed the low leakage of
protons from the reconstituted liposomes. The mixture was incubated at
40 °C and preilluminated for 15 min before the ATP synthesis
reaction was started by addition of 2 mM MgSO4.
Aliquots of the illuminated sample were taken at different reaction
times, and the reactions were quenched with trichloroacetic acid. The
ATP content was measured using a luciferin-luciferase assay.
 |
RESULTS |
Time-dependent Change of the ATPase Activity of
V1-ATPase--
Fig.
1a shows the time course of
ATP hydrolysis measured with the ATP regenerating system. Hydrolysis of
200-4000 µM ATP by V1-ATPase proceeded in
three distinct phases. Within 10 s after the reaction was started
by addition of enzyme, an apparent short initial lag was observed (Fig.
1a). The initial lag phase rapidly transformed to a second
phase of high rate of hydrolysis. Then, the rate of hydrolysis was
decelerated slowly. The ATP hydrolysis almost stopped after 20 min
(data not shown). Fig. 1b shows the change of absorbance at
340 nm over time (dA340/dt), illustrating
the three phases of hydrolysis. The decrease in dA340/dt corresponds to the activation of the ATPase and the increase to the
inactivation. The results indicate that the initially inactive species
of the V1-ATPase were rapidly activated, and the activated species were re-inactivated during ATP hydrolysis.

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Fig. 1.
ATP hydrolysis by V1- or
V0V1-ATPase. a, ATP hydrolysis
was monitored at 25 °C in the presence of an ATP regenerating
system; see under "Experimental Procedures." The reactions were
started by the addition of 10 µl of 5 µM enzyme
solution to 1.2 ml of reaction mixture containing the indicated
concentrations of ATP-Mg. b, the changes of the absorbance
at 340 nm in time (dA340/dt) was
calculated and plotted against the reaction time. c, ATP
hydrolysis by VoV1-ATPase. The reaction mixture
for VoV1-ATPase contained 0.05% Triton X-100.
Other conditions were the same as above.
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Interestingly, both the rates of initial activation and subsequent
inactivation of the V1-ATPase were dependent on the ATP concentration in the assay mixture. As shown in Fig. 1a, the
rapid inactivation was only observed at high concentrations of ATP. The
inactivation almost disappeared and the rate of ATP hydrolysis was
almost constant during turnover at 30 µM ATP (Fig. 1,
a and b, trace 0.03 mM). The
VoV1-ATPase also exhibited similar activation and inactivation phases in hydrolyzing 0.01-4 mM ATP-Mg
(Fig. 1c). The time-dependent changes were
analyzed using a simple sequential model for the activation and
inactivation processes, and the apparent first order rate constants for
each process were calculated by nonlinear regression fitting. The
apparent rate constants were plotted against the ATP concentrations. As
shown in Fig. 2, the rate constants of
inactivation exhibited a monophasic dependence on ATP concentration,
and the half-maximum rate of inactivation was attained at 140 µM ATP. The associated maximum inactivation rate constant
was 0.0055 s
1. We calculated the ATP concentration for
the half-maximum rate of activation with the same method. It was 70 µM ATP, and the associated maximum activation rate
constant was 0.65 s
1 (Fig. 2b).

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Fig. 2.
Correlation between the rate of inactivation
and activation of the V1-ATPase and the ATP concentration
in the assay mixture. The rates of inactivation and activation
were estimated by nonlinear curve fitting assuming sequential
activation and inactivation during the ATPase assay. Inset,
corresponding Eadie-Hofstee plots. a, the rate of
inactivation was plotted as a function of the ATP concentration. The
range of ATP concentrations was 40-4000 µM. The
solid line represents the best fit. b, the rate
of activation was plotted as a function of the ATP concentrations. The
solid line represents the best fit.
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By analogy to the inactivation of F1-ATPases (24, 31, 32),
we suspected that the inactivation was caused by entrapment of
inhibitory ADP at a catalytic site. To investigate the effect of ADP
for the inactivation, we assayed the ATPase activity of V1-ATPase by the measurement of inorganic phosphate with or
without the ATP regenerating system. When 50 µg/ml pyruvate kinase
and 2 mM phosphoenolpyruvate were present in the assay
mixture, the hydrolysis of ATP proceeded linearly up to 4 min after
addition of enzyme. Then, the rate of hydrolysis gradually decelerated 5 min after the reaction was started. On the other hand, in the absence
of the ATP-regenerating system, the deceleration of the rate of
hydrolysis occurred more rapidly, and the hydrolysis completely stopped
at 6 min (Fig. 3a). The
apparent rate constant of inactivation of the V1-ATPase
under these conditions was about 3 × 10
3
s
1 in the presence of an ATP-regenerating system, and
about 7 × 10
3 s
1 in the absence of an
ATP regenerating system. These values are in the same order of
magnitude as the value deduced in Fig. 2a. Fig.
3b shows the effect of ADP in the assay mixture on the rate of hydrolysis of ATP. Various amounts of ADP were added to the assay
mixtures in the absence of a regenerating system, and the hydrolysis of
4 mM ATP was assayed. Increasing ADP concentrations in the
assay mixtures led to a decrease of the rate of ATP hydrolysis.

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Fig. 3.
Effect of ADP-Mg for the inactivation.
a, the time course of Pi liberation by the
V1-ATPase in the absence (open circles) or
presence (solid circles) of the ATP regenerating system. The
reactions were started by the addition of 10 µl of 5 µM
enzyme to 490 µl of assay mixture containing 4 mM ATP. In
the presence of the ATP regenerating system, the assay mixtures
contained 50 µg/ml pyruvate kinase and 2 mM
phosphoenolpyruvate. The reactions were stopped at the indicated times,
and the generated Pi was measured by the
Pi-molybdate assay. b, time course of
Pi generation in the presence of various ADP-Mg
concentrations in the assay mixture. The reactions were started by the
addition of enzyme to the assay mixtures. The reactions were stopped at
indicated times, and the Pi in the assay mixture was
measured. , 0 µM ADP-Mg; , 10 µM;
, 40 µM; , 80 µM; , 200 µM; , 500 µM. c, inhibition
of the ATPase activity of the V1-ATPase by preincubation
with ADP-Mg. The V1-ATPase (1 µM) was
preincubated with the indicated concentrations of ADP-Mg for 120 min at
25 °C. Then, 20 µl were added into 1.2 ml of the ATP assay mixture
containing 4 mM ATP (see under "Experimental
Procedures").
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Furthermore, 1 µM V1-ATPase was preincubated
with various concentrations of ADP-Mg for 120 min at 25 °C, and the
residual ATPase activities were measured in the presence of 4 mM ATP-Mg. As shown in Fig. 3c, the extent of
inactivation of the V1-ATPase was dependent on the
concentration of added ADP-Mg.
These results strongly suggest that the mechanism of inactivation is
similar to that of the ADP-Mg inhibition observed for F1-ATPase.
Kinetics of ATP Hydrolysis by V1-ATPase--
Because
the ATP hydrolysis by V1-ATPase did not proceed linearly
over time, it is difficult to define the rate at a given ATP
concentration. In the present study, we plotted the maximum rate of ATP
hydrolysis at each ATP concentration (Fig.
4). The ATP concentration ranged from 40 to 4000 µM. The kinetic data were also plotted in V/S
versus V form (inset, Eadie-Hofstee plot). From
this plot, an apparent Km of 240 ± 24 µM and an apparent Vmax of
5.2 ± 0.5 units/mg were deduced. An apparent negative
cooperativity that is frequently observed for
FoF1- or F1-ATPase (31, 33-41, 43)
was not observed for the V1-ATPase here.

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Fig. 4.
Maximum rate of ATP hydrolysis by the
V1-ATPase at various concentrations of ATP. The rates
of hydrolysis of 40-4000 µM ATP were measured using 50 pmol of the V1-ATPase in the presence of the
ATP-regenerating system. The maximum rates of hydrolysis were
calculated with a computer program from the monitored time course data
and plotted against ATP concentration. Inset, Eadie-Hofstee
plots. The solid line represents the best fit.
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Binding of ATP to the Isolated Subunits--
The kinetics features
of inactivation of V1-ATPase during the ATPase reaction
described above are similar to those recently reported for the mutant
F1-ATPase, which lacks nucleotide binding at the
noncatalytic sites (32). In that case, turnover-dependent inactivation was explained as the failure to recover from the ADP
inhibited state due to the inability of nucleotide binding at
noncatalytic sites. If the V1-ATPase lacks nucleotide
binding to the noncatalytic subunit (subunit B), the similarity of
V1-ATPase to mutant F1-ATPase is well
understood. The lack of Walker motifs of B subunit of
V1-ATPase further underlined this idea. Thus, in an attempt
to characterize nucleotide binding to the isolated subunits of
V1-ATPase, we constructed over expression systems for the A
and B subunits and purified the products. We could successfully obtain
large amounts of these subunits (Fig. 5).
The isolated recombinant A and B subunits migrated as single protein
bands with little contamination.

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Fig. 5.
Analysis of the isolated A and B subunits by
SDS-PAGE. The samples were subjected to SDS-PAGE (15% acrylamide
gels), and proteins were stained with Coomassie Brilliant Blue R-250.
Lane 1, cell lysate of E. coli harboring pATPA
(50 µg); lane 2, cell lysate of E. coli
harboring pATPB (50 µg); lane 3, V1-ATPase (5 µg); lane 4, V1-ATPase (10 µg); lane
5, a subunit purified from cell lysate of E. coli
harboring pATPA (15 µg); lane 6, B subunit purified from
cell lysate of E. coli harboring pATPB (15 µg).
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Difference absorption spectra have been used to probe the binding of
adenine nucleotides to the F1-ATPase and its isolated subunits (29). When these proteins bind ADP or ATP, difference spectra
are induced by a red shift of the absorption maximum accompanied by a
slight decrease of the magnitude. The difference absorption spectra
induced by the interaction of ATP with the isolated A or B subunits
showed significantly different profiles (Fig.
6). Upon addition of ATP-Mg to the
isolated A subunit, a trough at 260 nm and a peak at 280 nm were
observed, and the magnitudes of the peak and the trough depended on the
amount of the added ATP. Saturation was observed when the concentration
of ATP reached about an equimolar concentration to the A subunit (Fig.
6a, inset). In contrast, neither a trough nor a
peak was observed for the B subunit (Fig. 6b).

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Fig. 6.
Absorbance difference spectra of the isolated
subunits at various ATP concentrations. 20-400 µM
ATP was added to 10 µM isolated A (a) or B
(b) subunit in the presence of 2 mM
MgCl2. Difference spectra were measured 5 min after mixing
all of the components (final concentration, 2 mM
MgCl2, 100 µM EDTA, and 20 mM
Tricine-OH, pH 8.0). Inset, the absorbance difference
A280-A260 is shown at
the indicated molar ratio of ATP per subunit. The horizontal
axis shows a molar ratio of ATP per subunit.
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2',3'-O-(2,4,6-Trinitrophenyl)-ATP is known to have a
significantly higher affinity for the nucleotide binding sites of
F1-ATPase and to give a clear difference spectrum in the
visible wavelength region upon binding (29, 42, 43). The difference
spectrum was induced by addition of
2',3'-O-(2,4,6-trinitrophenyl)-ATP to the isolated A
subunit, but was not induced in case of the B subunit (data not shown).
These results suggest that the isolated A subunit can bind 1 mol of
ATP-Mg per enzyme, but the isolated B subunit cannot bind ATP-Mg. The
ability and inability of ATP binding of the A and B subunit are
consistent with the presence and absence of the Walker motifs in these
subunits.
Analysis of Bound Nucleotide of V1-ATPase--
To
analyze bound nucleotide, the V1-ATPase was preincubated
with or without 1 mM ADP-Mg for 60 min, and free
nucleotides were removed with a Sephadex G-50 centrifuge column. The
enzyme was denatured with perchloric acid, and the amount of released
adenine nucleotide was quantified by anion exchange high performance
liquid chromatography. The analysis revealed that endogenous ADP on the purified V1-ATPase preparation was less than 0.1 mol per
mol of enzyme, whereas ADP-Mg-preincubated V1-ATPase
contained 1.5 mol of ADP per mol of the enzyme.
T. thermophilus VoV1-ATPase Synthesizes ATP
Coupled to Proton Flux--
ATP synthesis is the physiological role of
T. thermophilus VoV1-ATPase (15). To
measure ATP synthesis, we co-reconstituted VoV1-ATPase with bR into proteoliposomes. A
light-induced pH gradient was generated, and the proton permeability
was monitored using the fluorescence of the pH-sensitive probe pyranine
trapped inside of the co-reconstituted
VoV1-ATPase-bR proteoliposomes. Light-induced proton translocation was observed and the accumulation of protons saturated after illumination for 10 min. Because the level of accumulated proton remained relatively constant after the illumination, the VoV1-ATPase-bR proteoliposomes have a low
proton permeability (Fig. 7,
inset).

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Fig. 7.
Light-driven ATP synthesis by
V0V1-ATPase-bR proteoliposomes. ATP
synthesis was started by the addition of 2 mM
MgSO4 to the assay mixture. The aliquots of 50 µl were
taken at indicated time, and 50 µl of 4% trichloroacetic acid were
added. After neutralization by the addition of 20 µl of 2 M potassium-phosphate buffer (pH 7.5), ATP content was
measured. a, shown are time courses of ATP synthesis in the
presence ( ) or absence ( ) of 20 µM carbonyl cyanide
p-trifluoromethoxyphenylhydrazone in the reaction mixture.
Inset, light-driven proton pumping of
V0V1-ATPase-bR proteoliposomes. b,
analysis of the VoV1-ATPase by 15% of
SDS-PAGE. Lane 1, molecular size standards (97, 66, 30, 45, 21, and 14 kDa); lane 2, 10 µg of V1-ATPase;
lane 3, 20 µg of
VoV1-ATPase.
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After preillumination for 15 min to generate a stable transmembrane pH
gradient, the ATP synthesis reactions were started by addition of
MgSO4 (indicated as time 0 in Fig. 7). The
synthesized ATP was measured with the luciferin-luciferase assay. ATP
synthesis proceeded with a constant rate for 40 min after starting the
reaction. The constant rate of ATP synthesis was found to be 0.67 µmol of ATP mg
1min
1, which is 3-4 times
larger than the one of FoF1-ATPase from
thermophilic bacterium, PS3 (30). The time-dependent
inhibition by ADP observed in case of ATP hydrolysis was not observed
in ATP synthesis.
 |
DISCUSSION |
Inactivation of V1-ATPase May Be Caused by Entrapment
of Inhibitory ADP-Mg in a Catalytic Site--
The results presented
clearly show three distinct phases of ATP hydrolysis by the
V1-ATPase in the presence of an ATP regenerating system
(Fig. 1, a and b). The initial lag apparently
shows the presence of initial inhibited species of
V1-ATPase. The inhibited enzyme changed rapidly to an
active form. The cause of this initial inhibition is not clear at
present. The activated enzyme was re-inactivated during turnover. The
apparent half-maximum rate of the inactivation was attained at 140 µM ATP, which reflects the affinity of the ATP binding
site for the inactivation. The presence of ADP-Mg in the assay mixture
increased the rate of turnover-dependent inactivation.
The specific activity of V1-ATPase is 5.2 unit/mg protein
at 25 °C in the presence of an ATP-regenerating system, but we
previously reported that the specific activity of V1-ATPase
was about 0.1 unit/mg protein at 55 °C in the absence of ATP
regenerating system (10). Probably, the low specific activity was due
to the turnover-dependent inactivation of the
V1-ATPase. In addition, contaminant ADP in the ATP solution
could increase the rate of inactivation, so that ATP hydrolysis almost
stopped 1-2 min after starting the reaction.
Previously, a similar slow inactivation of yeast V-ATPase was reported
by Kibak et al. (44). They showed sulfite eliminates the
inactivation, but in our case, 33 mM
Na2SO3 apparently inhibited the initial rate of
the ATP hydrolysis of T. thermophilus V1-ATPase (data not shown).
In the case of mitochondorial F1-ATPase, Jault and Allison
(34) indicated that three kinetics phases were present at low ATP
concentration. An initial burst phase decelerated rapidly to a slow
intermediate phase, which, in turn, gradually accelerated to a final
steady-state rate. They postulated that the transition of the initial
burst phase to the slow intermediate phase was caused by accumulation
of inhibitory ADP-Mg at a catalytic site and that the transition of the
intermediate phase to the final steady state was caused by binding of
ATP to noncatalytic sites, which promoted the dissociation of
inhibitory ADP-Mg from the affected catalytic site (31, 34). Recently,
Matsui et al. (32) observed a rapid and nearly complete
turnover-dependent inactivation of a mutant
F1-ATPase, which lacks the ability of nucleotide binding at
noncatalytic sites. The mutant F1-ATPase was also
completely inactivated by prior incubation with stoichiometric ADP-Mg;
thus, they concluded that the entrapment of ADP-Mg in a catalytic site
caused the turnover-dependent inactivation. Interestingly, they also observed the ATP concentration dependence of the rate of
inactivation and found that the half-maximum rate of inactivation was
attained at 5 µM ATP, which coincides with one of the two Km values, about 4 µM, obtained from
initial rate analysis (32). In this study, we observed a similar
inactivation of V1-ATPase during turnover of ATP
hydrolysis. The Km of 240 µM
determined for the hydrolysis of 40-4000 µM ATP may be
comparable to the apparent Kd of 140 µM for inactivation. Furthermore, nearly complete
inactivation of V1-ATPase was attained by prior incubation of V1-ATPase with ADP.
The analogy of the inactivation of F1-ATPases leads us to
postulate that the inactivation of V1-ATPase is caused by
the entrapment of inhibitory ADP-Mg on the catalytic sites. The role of
the noncatalytic nucleotide binding sites of F1-ATPase
during ATP hydrolysis is the release of inhibitory ADP bound at the
catalytic sites. It was shown that a mutant of F1-ATPase
that lacked the ability of nucleotide binding on the noncatalytic sites
exhibited strong inhibition by ADP (32). The strong inactivation of the
V1-ATPase seems similar to that of the mutant of
F1-ATPase. Actually, the B subunit of the
V1-ATPase does not have a region homologous to the Walker
motifs A and B. As expected, the interaction of ATP and the isolated B
subunit was not observed in measurements of difference spectra. Taken
together, we prefer the view that the noncatalytic B subunit in the
T. thermophilus V1-ATPase does not bind
nucleotide or has a only very weak affinity for nucleotides. The
results obtained suggest that the inactivation of V1-ATPase was due to the failure of binding of ATP to noncatalytic sites.
Endogenous ADP on V1-ATPase was less than 0.1 mol per mol
of enzyme, whereas 1.5 mol of ADP per mol of the enzyme was bound to
V1-ATPase after the preincubation with 1 mM
ADP-Mg for 1 h. Because the loaded nucleotides could not be
removed by centrifugation elutions, this nucleotide is thought to bind
the catalytic site with high affinity.
The mechanism of initial activation of V1-ATP is unknown.
Unlike the irreversibly turnover-dependent inactivated
enzyme, the initial inhibited enzyme is rapidly activated by the
binding of ATP. Furthermore, the initial activation occurred at a lower
ATP concentration range than the turnover dependent inactivation. These
results clearly show that the initial inhibited form is not identical
to the irreversible inactivated form. Further studies will be necessary
to clarify the characteristics of the bound adenine nucleotides and the
initial inhibited form.
VoV1-ATPase Can Synthesize ATP in
Co-reconstituted Liposomes--
Several findings indicate that the
physiological role of VoV1-ATPases in some
archea and T. thermophilus is the synthesis of ATP coupled
to a proton flux (7-10, 13, 15, 25, 26). The results in this study
give direct evidence for the ability of T. thermophilus
VoV1-ATPase to synthesize ATP coupled to proton flux. This is the first report of ATP synthesis with a reconstituted proteoliposome of a VoV1-ATPase. We used the
VoV1-ATPase-bR co-reconstituted proteoliposomes
because a steady pH potential is attained by light-induced proton
pumping. The reaction mixture for ATP synthesis contained 2 mM ADP-Mg, which is sufficient to induce complete
inactivation of the VoV1-ATPase for ATP
hydrolysis. However, ATP synthesis continued for up to 40 min indicates
that ADP-Mg-induced inactivation does not occur under ATP synthesis
conditions. It is possible that the membrane potential and/or the pH
gradient protects VoV1-ATPase from ADP-Mg
inactivation.
The particular characteristics of T. thermophilus
VoV1-ATPase, where ATP
hydrolysis-turnover-dependent inactivation occurs under ATP
hydrolysis condition but ATP synthesis is not inhibited in the presence
of a proton motive force, are thought to be favorable for physiological
ATP synthesis. When the proton motive force is close to zero for the
cell, the hydrolysis of intracellular ATP may be inhibited by the
generation of the inactivated species of the
VoV1-ATPase, so that a rapid decrease of
intracellular ATP is avoided.
During the preparation of this report, an interesting paper by Bald
et al. was published (45). They reconstituted the mutant FoF1-ATPase, which lacks nucleotide binding to
the noncatalytic site, into liposomes with bR and examined light-driven
ATP synthesis. Contrarily to the quickly inhibition of ATP hydrolysis
activity, the mutant FoF1-ATPase synthesized
ATP at nearly constant rate up to 60 min. This results further
reinforces the similarity of the VoV1-ATPase to
the mutant FoF1-ATPase, which lacks nucleotide binding to the noncatalytic subunit.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Masao Chijimatsu and Masafumi
Odaka of the Riken Institute for the quantitative total amino acid
analysis of V1-ATPase, and we thank Mr. Shibata and Dr.
Hisabori for stimulating discussion and Michael Stumpp for carefully
reading the manuscript.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed. Fax: 81-76-234-4493;
E-mail: yokoken{at}kenroku.kanazawa-u.ac.jp.
The abbreviations used are:
bR, bacteriorhodopsin; PAGE, polyacrylamide gel electrophoresis.
 |
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