(Received for publication, May 4, 1995; and in revised form, December 8, 1995)
From the
The H,K
-ATPase of intact
gastric vesicles has two K
values for ATP
hydrolysis, 7 and 80 µM. Irradiation of vesicles with
ultraviolet light in the presence of 1 mM ATP resulted in
K
-ATPase activity that shows only the low affinity ATP
binding. The irradiation stimulated or inhibited proton uptake rate
compared with control vesicles at high or low ATP concentrations,
respectively. The relation between proton uptake rate and
K
-ATPase activity at different ATP concentrations was
linear with irradiated vesicles and nonlinear with control vesicles.
These results indicate that hydrolysis at the high affinity ATP binding
site regulates the energy-transport coupling in negative and positive
manners at high and low ATP concentrations, respectively. The complete
inhibition of K
-ATPase by a specific proton pump
inhibitor E3810 (rabeprazole)
(2-{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulfinyl}-1H-benzimidazole
sodium salt) occurred when E3810 bound to half of the
-subunit of
H
,K
-ATPase in unirradiated vesicles
at both 200 and 10 µM ATP, whereas the complete inhibition
of proton uptake occurred when E3810 bound to half or a quarter of the
-subunit at 200 or 10 µM ATP, respectively. These
results suggest that dimeric interaction between the
-subunits is
necessary for the enzyme activity at all ATP concentrations and that
dimeric or tetrameric interaction is necessary for proton transport at
high or low ATP concentrations, respectively.
The H,K
-ATPase is involved in
the final step of gastric acid secretion and secretes H
in exchange for
K
(1, 2, 3, 4, 5, 6) .
The gastric H
,K
-ATPase is classified
as a P-type ATPase, closely related to the
Na
,K
-ATPase(7) . The
H
,K
-ATPase consists of
- and
-subunits. The
-subunit is a catalytic subunit, and the
-subunit is suggested to maintain integrity of the enzyme
structure and affect ATP hydrolyzing activity(8) . The
H
,K
-ATPase has high and low K
values for ATP hydrolysis(3) .
Several different explanations are possible for the presence of high
and low K
values(9) . One
possible explanation is that the oligomeric interaction of
(
)-protomer results in two different affinities for ATP.
Diffraction pattern analysis of the electron microscopic image of
two-dimensional crystals of the enzyme provided evidence that the
H
,K
-ATPase exists as a dimer (10) or tetramer (11) of the
-subunit. So far,
there is no information as to whether close
-
-subunit contact
and interaction are necessary for the enzyme activity and active
transport of proton and potassium. In
Na
,K
-ATPase, specific cytoplasmic
regions of the
-subunit have been shown to be necessary for
-
contact(12, 13, 14) , and the
-subunit does not associate with
H
,K
-ATPase
-subunit(12, 13) .
In this paper, we studied
the biochemical basis of two K values for
ATP hydrolysis by gastric H
,K
-ATPase
and found that ATP hydrolysis at the high affinity site regulated
proton uptake with negative and positive cooperativity at high and low
ATP concentrations, respectively. Furthermore, from measurements of the
relations between ATP hydrolysis, proton transport rate and the amount
of specific binding of a proton pump inhibitor E3810, (
)we
found that at high ATP concentrations the functions of ATP hydrolysis
and proton transport require the dimeric interaction between
-subunits, but at low ATP concentrations, the function of ATP
hydrolysis requires the dimeric interaction, whereas that of proton
uptake requires the tetrameric interaction.
For determination of the ratio
of bound H,K
-ATPase inhibitor, E3810,
to the
-subunit of H
,K
-ATPase,
gastric vesicles were specifically labeled with
[
-methylene-
C]E3810. For specific
labeling, gastric vesicles (1 mg/ml) were incubated with 2 mM ATP in a solution containing 2 mM MgCl
, 150
mM KCl, 10 µg/ml valinomycin, and 40 mM Tris/HCl
(pH 7.40) for 20 min in the presence of various concentrations of
[
-methylene-
C]E3810. Reaction was
stopped by gel filtration through a Sephadex G-50 column equilibrated
with 150 mM KCl and 40 mM Tris/HCl (pH 7.40). The gel
filtration dissipated the proton gradient across the vesicle membrane,
resulting in decomposition of the activated E3810, and unreacted
[
-methylene-
C]E3810 was removed.
After the redetermination of protein concentration of the filtrate by
means of absorbance at 260 nm, the amount of bound
[
-methylene-
C]E3810/mg of vesicle
protein was determined with a liquid scintillation counter.
K
-ATPase and proton uptake activities of E3810-labeled
vesicles were also measured at low (10 µM) and high (200
µM) ATP concentrations. The
-subunit of
H
,K
-ATPase comprised 48.3% of the
total vesicle protein determined by densitometry of SDS-PAGE gels
stained with Coomassie Brilliant Blue R-250.
Figure 1:
Double-reciprocal plots between
[ATP] and K-ATPase activity of the control
and UV light-irradiated vesicles. Gastric vesicles (1 mg of protein/ml)
were irradiated with UV light for 1 h at 25 °C in the presence of 1
mM ATP (
) in the presence of valinomycin.
K
-ATPase activities were measured at various ATP
concentrations as described under ``Materials and Methods.''
For the control experiment, gastric vesicles were incubated without UV
light irradiation for 1 h at 25 °C (
). For another control
experiment, K
-ATPase activity of unirradiated vesicles
was measured in the presence of 15 mM KCl plus 135 mM choline chloride and nigericin in replacement of 150 mM KCl and valinomycin (
). Values are means ± S.E.
from three experiments. Straight lines were drawn using the V
and K
values
obtained by nonlinear least-square curve-fitting to the
Michealis-Menten equation.
Figure 2:
Effects of UV light irradiation on
K-ATPase activity in the presence and absence of ATP.
Gastric vesicles (1 mg of protein/ml) were irradiated with UV light at
260 nm for 1 h in the presence (
) and absence (
) of 1 mM ATP. For the control experiment (
), gastric vesicles were
incubated without UV light irradiation for 1 h at 25 °C. Then,
K
-ATPase was measured as a function of the ATP
concentration. Values are means ± S.E. from three
experiments.
Figure 3:
Effects of UV light irradiation on the
rate of proton uptake. Gastric vesicles (1 mg of protein/ml) were
irradiated with UV light for 1 h at 25 °C in the presence of 1
mM ATP. Proton uptake was determined from fluorescence quench
of acridine orange. A, each point represents the mean of three
observations of the initial slope of the fluorescence quench. The
difference between the UV light-irradiated () and control vesicles
(
) are significant (*, p < 0.05,**, p <
0.01, unpaired Student's t test) at 5 and 10 µM ATP. B, the values represent means of nine observations
of the initial slope of fluorescence quench at 500 µM ATP.
The difference of the rates of proton uptake between control and UV
light-irradiated vesicles is significant (* p < 0.05,
unpaired Student's t test). Proton uptake was measured
in a solution containing 10 µg/ml of gastric vesicles, 150 mM KCl, 40 mM Tris/HCl (pH 7.40), 2 mM MgCl
, 10 µg/ml of valinomycin, 100 µM AMP, 5 µM acridine orange, 0.8 mM phosphoenolpyruvate, 4 units/ml pyruvate kinase, and various
concentrations of ATP at 25 °C.
The relationship between K-ATPase activity and
proton uptake rate was not linear in control vesicles, but it was
linear in UV light-irradiated vesicles (Fig. 4), suggesting that
ATP hydrolysis at the high affinity ATP binding site regulates the
coupling between K
-ATPase activity and proton uptake
rate in negative and positive cooperative manners at high and low ATP
concentrations, respectively.
Figure 4:
Correlation between the
K-ATPase activity and the rate of proton uptake of UV
light-irradiated vesicles. Gastric vesicles (1 mg of protein/ml) were
irradiated with UV light for 1 h at 25 °C in the presence of 1
mM ATP (
). Then, K
-ATPase activity and
the rate of proton uptake were measured. For the control experiment
(
), gastric vesicles were incubated without UV light irradiation.
Each point represents the mean of three observations of
K
-ATPase activity and proton uptake rate. The
regression value of the linear fit (
) was
0.9949.
Figure 5:
Specificity of E3810 binding to
-subunit of H
,K
-ATPase. Gastric
vesicles (1 mg of protein/ml) were incubated with 5 µM of
radioactive E3810 in the presence of 150 mM KCl, 40 mM Tris/HCl (pH 7.40), 2 mM MgCl
, 10 µg/ml
valinomycin, and 2 mM ATP for 20 min at room temperature.
Immediately, the sample solution was applied to SDS-PAGE (top). The radioactivities of sliced gel pieces were measured
as described under ``Materials and Methods'' (bottom).
Figure 6:
Relationship between the amount of bound
E3810 and the K-ATPase activity (A) and
between the amount of bound E3810 and the rate of proton uptake (B) of gastric vesicles measured at 200 µM ATP.
Gastric vesicles (1 mg of protein/ml) were incubated with various
concentrations of radioactive E3810 in the presence of 150 mM KCl, 40 mM Tris/HCl (pH 7.40), 2 mM MgCl
, 10 µg/ml valinomycin, and 2 mM ATP
for 20 min at room temperature. The reaction was terminated by gel
filtration through Sephadex G-50 column equilibrated with 150 mM KCl and 40 mM Tris/HCl (pH 7.40). The amount of bound
E3810 was measured with a liquid scintillation counter.
K
-ATPase activity was measured at 200 µM ATP. The rate of proton uptake was determined by the initial slope
of fluorescence quench of acridine orange after the addition of 200
µM ATP in a solution containing 10 µg/ml of vesicles,
150 mM KCl, 40 mM Tris/HCl (pH 7.40), 2 mM MgCl
, 10 µg/ml valinomycin, 100 µM AMP, 5 µM acridine orange, 0.8 mM phosphoenolpyruvate, and 3 units/ml pyruvate kinase at 25 °C.
The experimental values less than 10% of the control were omitted for
the linear least-square fits, which gave the intercepts of 2.6 (A) and 2.1 nmol/mg of protein (B) at the complete
inhibition of the enzyme activity and proton uptake,
respectively.
Figure 7:
Relationship between the amount of bound
E3810 and the K-ATPase activity (A) and
between the amount of bound E3810 and the rate of proton uptake (B) of gastric vesicles measured at 10 µM ATP.
These experiments were carried out as described in the legend to Fig. 6. K
-ATPase activity and proton uptake
were measured at 10 µM ATP. The experimental values less
than 10% of the control were omitted for the linear fits, which gave
the intercepts of 2.5 (A) and 1.0 nmol/mg of protein (B) at the complete inhibition of the enzyme activity and
proton uptake, respectively.
The ratio of bound E3810/-subunit was
calculated taking into consideration that E3810 binds only to the
-subunit of H
,K
-ATPase, the
-subunit comprised 48.3% vesicle proteins and the mass of the
-subunit is 114 kDa; for example, 2.1 nmol/mg of proteins gives
the ratio of 0.50 (= 2.1
10
1.14
10
/(10
0.483)). Table 2shows the ratios necessary for complete inhibitions of
K
-ATPase activity and proton uptake at 200 and 10
µM ATP. These results suggest that binding of one molecule
of E3810 can arrest the K
-ATPase activity of two
molecules of the
-subunit at the both high and low ATP
concentrations, and it can arrest protein uptake of two or four
molecules of the
-subunit at the high or low ATP concentration,
respectively.
The gastric H,K
-ATPase
consists of
- and
-subunits. Crystallization of
H
,K
-ATPase has shown that two (9) or four (10) monomers comprised the crystalline
unit, which may suggest the presence of
-
-subunit contact and
interaction, as shown in
Na
,K
-ATPase(12, 13, 14) .
So far there has been no report whether the
-
-subunit
interaction of H
,K
-ATPase is involved
in the function of the enzyme activity and the active ion transport.
The present study using a specific proton pump inhibitor E3810 has
demonstrated that the ratio of bound E3810/
-subunit necessary for
the complete inhibition of K
-ATPase activity is about
0.6 at both high and low ATP concentrations, and the ratio necessary
for the complete inhibition of proton uptake is 0.5 or 0.24 at high or
low ATP concentrations, respectively (Table 2). That is, when
E3810 binds to one of two
-subunits, both subunits lose the
catalytic activity at every ATP concentration. When E3810 binds to one
of two
-subunits, both subunits lose the proton transporting
activity at high ATP concentration, and when E3810 binds to one of four
-subunits, all four subunits lose the proton transporting activity
at low ATP concentrations. These results strongly suggest that the
functional unit for K
-ATPase activity is the
(
)
-diprotomer at every ATP concentration, whereas
the functional unit for proton transport is
(
)
-diprotomer at high ATP concentrations and the
(
)
-tetraprotomer at low ATP concentrations.
When H,K
-ATPase was irradiated by
UV light in the presence of ATP, the high affinity ATP binding was
inhibited, whereas the low affinity ATP binding remained intact (Fig. 1). In these irradiated vesicles, proton uptake rate was
stimulated at high ATP concentrations and inhibited at low ATP
concentrations compared with those of control vesicles (Fig. 3).
Furthermore, control vesicles show negative or positive cooperative
coupling between the K
-ATPase activity and proton
uptake rate at high or low ATP concentrations, respectively, whereas
the UV light-irradiated vesicles show no cooperativity (Fig. 4).
These results suggest that the high affinity ATP binding site has a
regulatory role in addition to that of ATP hydrolysis.
The presence
of AMP-PNP enhanced K-ATPase activity in control
vesicles but decreased it in UV light-irradiated vesicles. We propose
the following explanation. Although AMP-PNP competes with ATP and
inhibits ATP hydrolysis at both ATP binding sites in control vesicles,
competition at the high affinity site diminishes the negative
cooperativity, resulting in a net increase of K
-ATPase
activity. In UV light-irradiated vesicles, which have only the low
affinity ATP binding site, AMP-PNP competition with ATP inhibits
K
-ATPase activity.
We suggest that the presence of
high and low affinity ATP binding sites in
H,K
-ATPase is due to the dimeric
-
interaction shown in this study. Other explanations,
however, are also possible as previously discussed for
Na
,K
-ATPase(9, 27) .
For example, solubilized
Na
,K
-ATPase protomer had two
different affinities for ATP(27) . We have shown that
H
,K
-ATPase has only one ATP binding
affinity (K
= 5 µM) when
measured in the presence of nigericin, which dissipates the proton and
potassium ion gradients across the vesicle membrane. This result shows
that each
-subunit has a single ATP binding site that is involved
in hydrolysis. Our explanation clearly differs from a previous one that
the nucleotide binds to H
,K
-ATPase
with different affinities in the sequential reaction cycle, E
ATP
H
and EP
H
ADP (or EP
H
ATP)(28) .
In
conclusion, we propose that the dimeric or tetrameric subunit
interaction is necessary for the function of
H,K
-ATPase, and two different
affinities for ATP of this enzyme are due to the dimeric subunit
interaction, which is sensitive to UV light irradiation and pH and/or
K
gradient across the vesicle membrane.