From the Department of Biochemistry, Institute of Cellular Signaling, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Both Na+,K+-ATPase and
H+,K+-ATPase belong to the family of P-type
ATPases, which transport ions across the plasma membrane (1). The
Na+,K+-ATPase is found in almost all animal
cells and is essential for the maintenance of cellular ion gradients,
whereas the gastric H+,K+-ATPase is located in
parietal cells of the gastric mucosa, where it is responsible for acid
secretion by the stomach. These
X+,K+-ATPases1 couple
ATP hydrolysis to countertransport of
X+ (Na+ or H+) and K+
as can be described by the Post-Albers scheme (2-4). Both ATPases consist of an Assembly of the Eventually the enzymes are transported either to the plasma membrane
(Na+,K+-ATPase) or to the tubulovesicles
(H+,K+-ATPase). Elegant studies by Caplan and
co-workers (24, 25) have localized the sorting signal of
Na+,K+-ATPase and
H+,K+-ATPase to a sequence of eight amino acids
present in the fourth predicted transmembrane domain of the Studies of the hybrid ATPase consisting of the
Na+,K+-ATPase Expression Constructs--
The rat gastric
H+,K+-ATPase Production of Recombinant Viruses--
Competent DH10bac
Escherichia coli cells (Life Technologies, Breda, The
Netherlands) harboring the baculovirus genome (bacmid) and a
transposition helper vector, were transformed with the pFastbacdual transfer vector containing different cDNAs. Upon transition between the Tn7 sites in the transfer vector and the bacmid, recombinant bacmids were selected and isolated (36). Subsequently, insect Sf9 cells were transfected with recombinant bacmids using
Cellfectin reagent (Life Technologies). After 3 days, the recombinant
baculoviruses were harvested and used to infect Sf9 cells at a
multiplicity of infection of 0.1. Four days after infection, the
amplified viruses were harvested.
Preparation of Membranes--
Sf9 cells were grown at
27 °C in 100-ml spinner flask cultures (9). For production of the
ATPases subunits 1.0-1.5 × 106 cells/ml were
infected at a multiplicity of infection of 1-3 in Xpress medium
(BioWittaker, Walkersville, MD) containing 1% ethanol (37) and
incubated for 3 days. The Sf9 cells were harvested by
centrifugation at 2,000 × g for 5 min, and resuspended
at 0 °C in 0.25 M sucrose, 2 mM EDTA, and 25 mM Hepes/Tris (pH 7.0). The membranes were sonicated twice
for 30 s at 60 W (Branson Power Co., Denbury, CT), after which the
disrupted cells were centrifugated at 10,000 × g for
30 min. The supernatant was recentrifugated at 100,000 × g for 60 min and the pelleted membranes were resuspended in
the above mentioned buffer and stored at Protein Determination--
Protein was determined with the
modified Lowry method described by Peterson (39) using bovine serum
albumin as a standard.
Western Blotting--
Protein samples from the membrane fraction
were solubilized in SDS-PAGE sample buffer and separated on SDS gels
containing 10% acrylamide according to Laemmli (40). For
immunoblotting, the separated proteins were transferred to Immobilon
polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The
Immunoprecipitation--
Immunoprecipitation was performed
essentially as described (45). Either 10 µg of monoclonal antibody
1F11 directed to the H+,K+-ATPase ATPase Activity Assay--
The ATPase activity was determined
with a radiochemical method (48). For this purpose 0.6-7 µg of
Sf9 membranes were added to 100 µl of medium, which contained
1 mM MgCl2, 0.2 mM EGTA, 0.1 mM EDTA, 1 mM NaN3, and 25 mM Tris-HCl (pH 7.0). For determination of
Na+,K+-ATPase activity, 100 µM
[ ATP Phosphorylation Capacity--
ATP phosphorylation was
determined as described (37). Sf9 membranes (6-70 µg) were
incubated at 0 °C in 25 mM Tris acetic acid (pH 6.0), 1 mM MgCl2 in a volume of 50 µl. For
phosphorylation of Na+,K+-ATPase, 100 mM NaCl was added to the incubation buffer. This phosphorylation is presented as the difference with and without 10 mM KCl. Phosphorylation of
H+,K+-ATPase is presented as the difference
with and without 0.1 mM SCH 28080. After 30-60 min
preincubation, 10 µl of 0.6 µM
[ Calculations--
The K0.5 value is
defined as the concentration of effector (K+) giving the
half-maximal activation and the IC50 as the value giving
50% inhibition of the maximal activation. Data are presented as mean
values with standard error of the mean. Differences were tested for
significance by means of the Student's t test.
Materials--
The cDNA clones of the
H+,K+-ATPase Recombinant baculoviruses expressing
Na+,K+-ATPase,
H+,K+-ATPase, their - and
-subunits of
Na+,K+-ATPase and
H+,K+-ATPase were expressed in Sf9 cells
in different combinations. Immunoprecipitation of the
-subunits
resulted in coprecipitation of the accompanying
-subunit independent
of the type of
-subunit. This indicates cross-assembly of the
subunits of the different ATPases. The hybrid ATPase with the catalytic
subunit of Na+,K+-ATPase and the
-subunit of
H+,K+-ATPase (NaK
HK
) showed an ATPase
activity, which was only 12 ± 4% of the activity of the
Na+,K+-ATPase with its own
-subunit.
Likewise, the complementary hybrid ATPase with the catalytic subunit of
H+,K+-ATPase and the
-subunit of
Na+,K+-ATPase (HK
NaK
) showed an ATPase
activity which was 9 ± 2% of that of the recombinant
H+,K+-ATPase. In addition, the apparent
K+ affinity of hybrid NaK
HK
was decreased, while the
apparent K+ affinity of the opposite hybrid HK
NaK
was
increased. The hybrid NaK
HK
could be phosphorylated by ATP to a
level of 21 ± 7% of that of
Na+,K+-ATPase. These values, together with the
ATPase activity gave turnover numbers for NaK
and NaK
HK
of
8800 ± 310 min
1 and 4800 ± 160 min
1, respectively. Measurements of phosphorylation of
the HK
NaK
and HK
enzymes are consistent with a higher
turnover of the former. These findings suggest a role of the
-subunit in the catalytic turnover. In conclusion, although both
Na+,K+-ATPase and
H+,K+-ATPase have a high preference for their
own
-subunit, they can function with the
-subunit of the other
enzyme, in which case the K+ affinity and turnover number
are modified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and a
-subunit, which assemble with a 1:1
stoichiometry to form a stable heterodimer. The catalytic
1-subunits of Na+,K+-ATPase and
H+,K+-ATPase share a high degree of identity
(63%), in contrast to their heavily glycosylated
1-subunits, which are structurally similar but only 30% identical.
- and
-subunits is important for conformational
stability of the functional holoenzyme (5, 6). This formation of a
complex between
- and
-subunits is also essential for enzyme
activity (7-9) and occurs before the subunits are transported from the
endoplasmic reticulum to the plasma membrane (10). Lemas et
al. (11) showed that the carboxyl-terminal 161 amino acids of the
Na+,K+-ATPase
-subunit are sufficient for
assembly with the
-subunit. More recently, Colonna et al.
(12) demonstrated that only four amino acids (SYGQ) in the
extracellular loop between the predicted transmembrane helixes 7 and 8 are crucial for
-
subunit interactions. These four evolutionary
conserved amino acids are also present in the
-subunit-binding
region Arg898-Thr928 of the
H+,K+-ATPase
-subunit (13). In addition,
Wang et al. (14) revealed that the
Na+,K+-ATPase
-subunit containing
Gln905-Val930 of the gastric
H+,K+-ATPase
-subunit (including SYGQ)
preferentially assembles with the H+,K+-ATPase
-subunit. Many investigators have maintained that the extracellular
domain as well as the cytoplasmic and transmembrane domains of the
-subunit are important for assembly with the
-subunit (13,
15-23).
-subunit
protein. In the cytoplasmic tail of the
H+,K+-ATPase
-subunit a functional
endocytosis signal was found. The presence of this motif accounts for
the returning of the pump from the apical cell membrane to its
intracellular storage compartment resulting in inactivation of acid
secretion (26).
-subunit and the
H+,K+-ATPase
-subunit have sought to analyze
the function of the
-subunit. This hybrid ATPase binds ouabain and
transports cations across the membrane (27, 28). The extracellular
region of the H+,K+-ATPase
-subunit is
probably responsible for the slightly higher apparent Na+
affinity and the lower apparent K+ affinity of this hybrid
compared with the Na+,K+-ATPase (20, 29). In
recent years, it has been demonstrated that the predicted transmembrane
segments 4, 5, and 6 are involved in cation occlusion (30, 31). The
-subunits of both enzymes probably participate in stabilizing this
occluded cation intermediate (32, 33). The question arises as to
whether the complementary hybrid, consisting of the catalytic subunit
of the H+,K+-ATPase and the
-subunit of the
Na+,K+-ATPase, also possesses catalytic
activity. We therefore measured both the
Na+,K+-ATPase activity and the
H+,K+-ATPase activity for both hybrid ATPases.
Furthermore, this study examines the possible role of
-subunits in
the apparent K+ affinity of both
K+-dependent ATPases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit cDNA (9) was
digested with BglII and ligated into the BamHI
site of the pFastbacdual vector (HK
) (Life Technologies, Breda, The
Netherlands). The
-subunit cDNA of the rat gastric
H+,K+-ATPase (9) was digested with
BamHI and ligated into the BbsI site of the
pFastbacdual vector containing the H+,K+-ATPase
-subunit (HK
). The rat Na+,K+-ATPase
1-subunit cDNA (34) HindIII fragment was
ligated into the HindIII site of the pFastbacdual vector
(NaK
, with a shortened multiple cloning site through a
BamHI-XbaI deletion) or into the pFastbacdual
vector (also with BamHI-XbaI deletion) containing the H+,K+-ATPase
-subunit (NaK
HK
). The
sheep Na+,K+-ATPase
1-subunit
cDNA (35) was digested with SmaI and SpeI and
ligated into the SmaI and NheI site of the
pFastbacdual vector containing the
Na+,K+-ATPase
1-subunit
(NaK
) or into the XhoI and NheI site of the pFastbacdual vector containing the H+,K+-ATPase
-subunit (HK
NaK
). All
-subunits were cloned downstream of
the polyhedrin promoter, and all
-subunits downstream of the p10
promoter. As mock, the baculovirus DZ1, only expressing
-galactosidase, was used (9).
20 °C. Native
H+,K+-ATPase and
Na+,K+-ATPase were isolated from the rat
gastric mucosa and the sheep kidney outer medulla, respectively (38).
These tissues were homogenized and centrifuged at 10,000 × g for 20 min at 4 °C. The supernatant was recentrifuged
at 100,000 × g for 30 min. The pelleted membranes were
resuspended in the above mentioned buffer and stored at
20 °C.
- and
-subunits of the gastric
H+,K+-ATPase were detected with the polyclonal
antibody HKB (41) and the monoclonal antibody 2G11 (42), respectively.
The
1- and
1-subunits of
Na+,K+-ATPase were detected with the polyclonal
antibody L25 (43) and the monoclonal antibody M17-P5-F11 (44),
respectively. The primary antibodies were detected using anti-mouse and
anti-rabbit secondary antibodies, which were labeled with peroxidase
(DAKO A/S, Denmark).
-subunit
(46) or 10 µl of the polyclonal serum L25 directed to the
Na+,K+-ATPase
-subunit (43) was added to 20 µl of protein A immobilized on agarose (50% (w/v), KemEnTec,
Copenhagen, Denmark) and resuspended in 500 µl of 10 mM
Tris-HCl (pH 8.0), 500 mM NaCl, and 0.05% Nonidet P-40
(Fluka, Bornem, Belgium). This mixture was incubated for 1 h at
4 °C with constant agitation, after which the protein A immobilized
on agarose was washed twice in the above mentioned buffer and once in
10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and
0.05% Nonidet P-40. Crude membrane proteins (~700 µg) were
solubilized in 500 µl of buffer containing 1% (w/v) octaethylene
glycol monododecylether (C12E8, Sigma), 10 mM Tris-HCl (pH 8.0), and 150 mM NaCl for
1 h at 4 °C. Following centrifugation at 10,000 × g for 5 min, the supernatant was incubated with the
antibodies bound to the protein A immobilized on agarose for 1 h
at 4 °C. The immunoprecipitates were collected by centrifugation for
a few seconds at 10,000 × g, washed 3 times in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and solubilized in SDS-PAGE sample buffer. After SDS-PAGE the ATPases subunits in the precipitates were identified by immunoblotting, using
the biotin-labeled antibodies L25 (43), M17-P5-F11 (44), and 2G11 (42).
The antibody HK9 was used for detection of the H+,K+-ATPase
-subunit (47). The primary
antibodies labeled with biotin were detected using streptavidin labeled
with peroxidase (Jackson ImmunoResearch, West Grove, PA), while HK9 was
detected with anti-rabbit secondary antibody, which was also labeled
with peroxidase (DAKO A/S, Denmark).
-32P]ATP, 100 mM NaCl, and 0.01 mM ouabain (in order to inhibit endogenous Na+,K+-ATPase from Sf9 cells) were
present. The specific activity is presented as the difference in
activity with and without 10 mM KCl. In the incubation
medium used for the measurements of the K+ activation curve
only 10 mM NaCl was present. For determination of
H+,K+-ATPase activity, 10 µM
[
-32P]ATP (specific activity 100-500 mCi
mmol
1), 1 mM KCl, and 0.1 mM
ouabain were present. The specific activity is presented as the
difference in activity with and without 0.1 mM SCH 28080. After incubation at 37 °C the reaction was stopped by adding 500 µl of 10% (w/v) charcoal in 6% (w/v) trichloroacetic acid and after
incubation at 0 °C the mixture was centrifuged for 30 s
(10,000 × g). To 200 µl of the clear supernatant,
containing the liberated inorganic phosphate
(32Pi), 4 ml of OptiFluor (Canberra Packard,
Tilburg, The Netherlands) was added and the mixture was analyzed by
liquid scintillation analysis. Blanks were prepared by incubating in
the absence of enzyme.
-32P]ATP was added and incubated for 10 s at
0 °C. The reaction was stopped by adding 5% trichloroacetic acid in
0.1 M phosphoric acid and the phosphorylated protein was
collected by filtration over a 0.8-µm membrane filter (Schleicher and
Schuell, Dassel, Germany). After repeated washing the filters were
analyzed by liquid scintillation analysis. Phosphorylation at different
ATP concentrations was performed at 21 °C and for
Na+,K+-ATPase in the presence of 20 µg/ml
oligomycin (a mixture of A, B, and C; Sigma).
- and
-subunits and the rat
and sheep cDNA clones of the Na+,K+-ATPase
1- and
1-subunits were provided by Drs.
G. E. Shull and J. B. Lingrel, respectively. Cellfectin,
competent DH10bac E. coli cells, and all enzymes used for
DNA cloning were purchased from Life Technologies Inc.
[
-32P]ATP (3000 Ci mmol
1) was purchased
from Amersham (Buckinghamshire, United Kingdom). SCH 28080, kindly
provided by Dr. A. Barnett, Schering-Plow, Kenilworth, NJ, was
dissolved in ethanol and diluted to its final concentration of 0.1 mM in 0.2% ethanol. The antibodies 2G11 and M17-P5-F11 were gifts of Drs. J. Forte (Berkeley) and W. J. Ball Jr.
(Cincinnati), respectively. Dr. J. V. Møller (Aarhus, Denmark)
provided antibody L25 and Dr. M. Caplan (Yale) antibodies HKB and HK9.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits, and their
hybrids were produced and Sf9
cells were infected. The membrane fractions of these Sf9 cells
expressing the recombinant proteins were isolated and Western blot
analysis revealed comparable expression patterns (Fig. 1). Both
Na+,K+-ATPase and
H+,K+-ATPase
-subunits detected with
antibodies L25 (43) and HKB (41), respectively, had an apparent
molecular mass of about 100 kDa. The antibody M17-P5-F11 (44)
visualized both a carbohydrate-free and a core-glycosylated form of the
Na+,K+-ATPase
-subunit. The monoclonal
antibody 2G11 (42) also recognized a carbohydrate-free and a
core-glycosylated form of the H+,K+-ATPase
-subunit. This carbohydrate-free form was of a similar molecular
mass as the carbohydrate-free Na+,K+-ATPase
-subunit, but the core-glycosylated
Na+,K+-ATPase
-subunit had a lower apparent
molecular mass, due to the presence of three glycosylation sites in
this subunit in contrast to the six glycosylation sites present in the
H+,K+-ATPase
-subunit.
View larger version (41K):
[in a new window]
Fig. 1.
Western blot of
Na+,K+-ATPase,
H+,K+-ATPase, and their hybrids. Membranes
(~10 µg) isolated from infected Sf9 cells were blotted and
the presence of Na+,K+-ATPase - and
-subunits was detected with antibodies L25 and M17-P5-F11,
respectively. The
- and
-subunits of
H+,K+-ATPase were detected with antibodies HKB
and 2G11, respectively. The subunits to which the different primary
antibodies were directed are indicated on the left and the
molecular weights are indicated on the right.
Interaction between the - and
-subunits in both
Na+,K+-ATPase and
H+,K+-ATPase is essential for a functionally
active enzyme. To examine the interaction between
- and
-subunits
in hybrid ATPases an immunoprecipitation assay was performed. The
-subunits of Na+,K+-ATPase and
H+,K+-ATPase were immunoprecipitated with
antibodies L25 (43) and 1F11 (46), respectively. In control experiments
both the Na+,K+-ATPase isolated from sheep
kidney and recombinant Na+,K+-ATPase were
precipitated with antibody L25. Similarly, rat gastric H+,K+-ATPase and recombinant
H+,K+-ATPase were precipitated by antibody
1F11. With the native enzymes only glycosylated
-subunits were
precipitated, with apparent molecular masses significantly higher than
those of their recombinant counterparts. This is due to the absence of
complex glycosylation in Sf9 cells. When the same method was
used for the hybrid ATPases, both
-subunits were also coprecipitated
with the other
-subunit (Fig. 2). This
indicates that there is not only an interaction between the
- and
-subunits of Na+,K+-ATPase and
H+,K+-ATPase, but also between the
- and
-subunits of the two hybrid ATPases. Although quantification in
these experiments is rather difficult, there seemed to be less
coprecipitated
-subunits in the hybrid ATPases than in the wild type
ATPases (NaK
, HK
).
|
When HKNaK
was present during immunoprecipitation with antibody
L25 (directed against the
-subunit of
Na+,K+-ATPase), the
-subunit of
Na+,K+-ATPase was unexpectedly precipitated.
Also when the H+,K+-ATPase
-subunit was
absent the Na+,K+-ATPase
-subunit was
precipitated (data not shown). In the absence of antibody L25 the
Na+,K+-ATPase
-subunit was not precipitated.
These findings suggest that the expressed
Na+,K+-ATPase
-subunit assembles with the
endogenous Na+,K+-ATPase
-subunit. Antibody
L25 apparently recognizes this endogenous
-subunit during
immunoprecipitation, but not on a Western blot. The absence of
unglycosylated
-subunit indicates that the assembly with the
endogenous
-subunit only occurred during the early stage of
infection, when the glycosylation machinery is still functional.
Na+,K+-ATPase activity was measured in the
presence of 100 µM ATP and optimal concentrations of
Na+ (100 mM) and K+ (10 mM). Because of the endogenous
Na+,K+-ATPase activity (50-100 nmol
mg1 protein h
1) present in the Sf9
membrane preparations, we determined the ouabain sensitivity for the
endogenously present Na+,K+-ATPase in mock
infected cells. The endogenous Na+,K+-ATPase
activity was completely inhibited at 1 × 10
5
M ouabain, while the recombinant activity was hardly
inhibited at this concentration. These findings are in line with those
of Lui and Guidotti (49). Using 1 × 10
5
M ouabain in the assay, we measured the recombinant ATPase
activity as the difference between the activity with and without 10 mM K+ (Fig.
3A). The activity of
NaK
HK
was 12 ± 4% (n = 3) of the activity
of NaK
. NaK
and mock infected cells did not show
Na+,K+-ATPase activity in the presence of
10
5 M ouabain.
|
In order to measure K+-stimulated
H+,K+-ATPase activity we used a suboptimal (10 µM) ATP concentration since at higher ATP concentrations the endogenous activity increases relatively more than the
H+,K+-ATPase activity. The K+
concentration (1 mM) used was optimal for
H+,K+-ATPase activity under these conditions
(50). The hybrid HKNaK
showed an SCH 28080-sensitive ATPase
activity which was 7 ± 1% (n = 3) of the
HK
activity (Fig. 3B). HK
and mock did not show any
SCH 28080-sensitive ATPase activity. In conclusion, these hybrids
possess only a fraction of the ATPase activity of
Na+,K+-ATPase or
H+,K+-ATPase when measured under similar conditions.
To determine if this low ATPase activity of the hybrid
ATPases could be due to changed ATP affinities, we measured
the ATPase activity at different ATP concentrations (0.03-1
mM). The results are given in a Woolf-Augustinsson-Hofstee
plot (Fig. 4). The intercept with the
y axis is equal to the maximal ATPase activity at infinite ATP concentrations. The maximal NaK ATPase activity was 1.8 ± 0.6 µmol mg
1 protein h
1, whereas the
activity for NaK
HK
was 0.21 ± 0.03 µmol mg
1
protein h
1 (which was 12 ± 4% of that of
NaK
, n = 3). The maximal HK
ATPase activity
was 1.4 ± 0.1 µmol mg
1 protein h
1,
whereas the activity for HK
NaK
was 0.12 ± 0.02 µmol
mg
1 protein h
1 (which was 9 ± 2% of
that of HK
, n = 3). The slope of this graph
represents the apparent ATP affinity. The apparent ATP affinities for
NaK
(197 ± 27 µM, n = 3) and
NaK
HK
(206 ± 5 µM, n = 3) are
similar. Also the apparent ATP affinities for HK
(17 ± 0.7 µM, n = 3) and HK
NaK
(13 ± 1.3 µM, n = 3) are not significantly different. This indicates that the lower ATPase activity of the hybrids
is not due to a change in ATP affinity.
|
Both Na+,K+-ATPase and
H+,K+-ATPase occlude K+ and
transport this ion across the plasma membrane. We compared the
K+ dependence of the overall ATPase activity of
Na+,K+-ATPase,
H+,K+-ATPase, and their hybrids. All ATPases
showed a biphasic activation curve, in which the maximal ATPase
activity was set at 100% (Fig. 5). The
increasing part of this curve is due to K+ activation of
the dephosphorylation step. The decreasing part is due to the
competition of K+ with H+ or Na+ at
the cytoplasmic activation sites. This directs the enzyme from the
E1 toward the E2
conformation, which cannot be overcome by the low ATP concentration
used. The K0.5 for K+ of NaK
was 0.5 ± 0.2 mM and the IC50 was 50 ± 9 mM (Fig. 5A). The curve of the hybrid
NaK
HK
was shifted to the right compared with the curve of
NaK
. The K0.5 and the IC50
values of this hybrid were slightly increased to 0.7 ± 0.3 mM (K0.5) and 103 ± 24 mM (IC50), respectively, when compared with
NaK
. In contrast, the K+ activation curve of
HK
NaK
was shifted to the left compared with the curve of HK
(Fig. 5B). Moreover, the K0.5 for
HK
(0.07 ± 0.01 mM) was decreased for the
hybrid HK
NaK
(0.02 ± 0.004 mM) and also the
IC50 was decreased from 10 ± 0.2 to 3.5 ± 0.7 mM. These shifts in K0.5 and
IC50 values were significant (p < 0.05, n = 3). Thus NaK
HK
had a slightly decreased
K+ affinity compared with NaK
, while the opposite
hybrid HK
NaK
had an increased K+ affinity compared
with HK
. These findings indicate that the
-subunits of both
Na+,K+-ATPase and
H+,K+-ATPase influence the K+
sensitivity of these enzymes.
|
The formation of an acid-stable phosphorylated intermediate during the
catalytic cycle is a characteristic property of the P-type ATPases.
Phosphorylation of Na+,K+-ATPase was measured
with Na+ present in the preincubation in order to shift the
equilibrium of the enzyme forms toward Na·E1.
NaKHK
was phosphorylated for 28 ± 11% compared with
NaK
(n = 3, Fig.
6A). Unlike phosphorylation of
NaK
HK
, phosphorylation of HK
NaK
was not visible, whereas HK
was normally phosphorylated (Fig. 6B). Changing the
temperature to 21 °C (31), longer incubation periods, inhibition
with K+ instead of SCH 28080, higher ATP concentrations, or
addition of triallylamine (51) still did not result in any measurable amount of phosphorylated intermediate. In order to determine the maximal phosphorylation level we measured the phosphorylation level at
different ATP concentrations (0.006-0.2 µM) at 21 °C. The data are plotted as the phosphorylation level versus the
ATP concentration (Fig. 7A)
and the same data are also visualized in a Woolf-Augustinsson-Hofstee
plot (Fig. 7B). In the last plot the apparent ATP affinity
and the maximal phosphorylation level can be determined more easily.
The ATP affinity is equal to the slope of the graph, while the
intercept with the y axis is equal to the maximal
phosphorylation level. For this interpretation it must be assumed that
the distribution of EP forms does not change over the range
of concentrations of ATP used. For HK
a maximal phosphorylation
level of 6.3 ± 0.9 pmol mg
1 protein with an
apparent ATP affinity of 23 ± 3 nM (n = 3) was measured. In the reaction mixture where the
Na+,K+-ATPase
-subunit was present
oligomycin was included, which increased the phosphorylation level by
about 30%. The apparent ATP affinities for NaK
(12 ± 2 nM, n = 3) and NaK
HK
(12 ± 1 nM, n = 3) are similar. However, the
maximal phosphorylation level for NaK
was 3.3 ± 1.0 pmol
mg
1 protein, whereas the maximal phosphorylation level
for NaK
HK
was 0.71 ± 0.10 pmol mg
1 protein
(which was 21 ± 7% of that of NaK
, n = 3).
These values, together with the maximal ATPase activity (determined at
infinite ATP concentrations) give turnover numbers for NaK
and
NaK
HK
of 8800 ± 310 min
1 and 4800 ± 160 min
1, respectively. These data are not corrected for the
suboptimal K+ concentration for the hybrid ATPase in the
ATPase reaction, which probably will increase the turnover number by
about 15%.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The -subunits of Na+,K+-ATPase and
H+,K+-ATPase are involved in correct folding
(10) of the
-subunits. In addition, the
-subunit of
H+,K+-ATPase plays a role in endocytosis of the
enzyme (24). Most probably, both
-subunits are also involved in the
modulation of the enzyme activity: Na+ and K+
affinities of the Na+,K+-ATPase were changed
when the Na+,K+-ATPase
-subunit was replaced
by the H+,K+-ATPase
-subunit (20, 29). In
the present study we demonstrate that the hybrid consisting of the
H+,K+-ATPase
-subunit and the
Na+,K+-ATPase
1-subunit has a
low ATPase activity with a high apparent K+ affinity and
probably a high turnover number as compared with H+,K+-ATPase.
Recombinant Na+,K+-ATPase,
H+,K+-ATPase, and their hybrids were produced
with the baculovirus expression system. The molecular mass of all
-subunits expressed was similar to that of the isolated
-subunits. However, the recombinant
-subunits were less
glycosylated than the isolated
-subunits, as has been reported
before (9, 52). Expression levels of the subunits of each hybrid ATPase were comparable to those of Na+,K+-ATPase and
H+,K+-ATPase, respectively.
Assembly of - and
-subunits is a crucial step in the formation of
active X+,K+-ATPases (7-9). The minimal
-subunit-binding site (12) in the
-subunits of
Na+,K+-ATPase and
H+,K+-ATPase are identical and therefore an
interaction between the
- and
-subunits of the different ATPases
seems likely. Both
-subunits are only 30% identical, but have a
high structural similarity. In the hybrid ATPase NaK
HK
the
subunits cross-assembled in a co-immunoprecipitation experiment. Others
also observed an interaction between the
Na+,K+-ATPase
-subunit and the
H+,K+-ATPase
-subunit (27, 28, 53). A
possible interaction between the H+,K+-ATPase
-subunit and the Na+,K+-ATPase
-subunit
has not been demonstrated so far. When in our experiments the
-subunit of the hybrid ATPase HK
NaK
was precipitated by
anti-Na+,K+-ATPase antibodies, the
-subunit
of Na+,K+-ATPase coprecipitated. However, for
both hybrid ATPases there seemed to be less coprecipitated
-subunits
compared with the wild type enzymes, indicating a less efficient
assembly between the different subunits of the hybrid ATPases. This
could be due to the 70% difference in amino acid composition or to the
difference in glycosylation between the two
-subunits.
Na+,K+-ATPase and
H+,K+-ATPase need hydrolysis of ATP to
transport cations across the membrane. Accordingly, functional hybrid ATPases should also have ATPase activity. We determined that the hybrid
ATPase NaKHK
possessed a low ATPase activity which was about 12%
of the activity of recombinant Na+,K+-ATPase.
Hybrid ATPase activity has not been measured so far, although in yeast
expressing the Na+,K+-ATPase
1-subunit and the H+,K+-ATPase
-subunit ouabain binding was observed (27). ATPase activity has been
measured in membranes isolated from yeast expressing the
Na+,K+-ATPase
1-subunit and a
chimeric
1-subunit (29). In oocytes expressing
NaK
HK
a small Na+,K+-pump current and
Rb+ uptake has been detected (28), although this could not
be confirmed by Ueno et al. (54), which might be due to the
very low activity of this hybrid ATPase. None of these assays were
performed for the hybrid ATPase HK
NaK
. When we measured the SCH
28080-sensitive ATPase activity, this hybrid exhibited also a low
ATPase activity, which was only 9% of the activity of recombinant
H+,K+-ATPase. Both hybrid ATPases have ATP
hydrolyzing activity, although this activity is much lower than that of
Na+,K+-ATPase and
H+,K+-ATPase. The ATP affinities of the hybrids
are similar to those of the wild type ATPases. So, the lower ATPase
activity of hybrid ATPases might result from a decreased amount of
functional hybrid ATPase molecules.
X+,K+-ATPases need K+ occlusion
before they can dephosphorylate (31) and probably the -subunit is
involved in this K+ occlusion (32, 33). When the
Na+,K+-ATPase
-subunit was replaced by the
H+,K+-ATPase
-subunit the K+
affinity in the ATPase reaction decreased similarly as has been reported in ouabain binding experiments by Eakle et al.
(20). However, the K+ affinity of the complementary hybrid
ATPase HK
NaK
was increased as compared with that of
H+,K+-ATPase. Thus the apparent K+
affinities of Na+,K+-ATPase and
H+,K+-ATPase are partly modulated through their
-subunits. In summary, the
-subunit of
Na+,K+-ATPase, as compared with the
-subunit
of H+,K+-ATPase, gives the enzyme a higher
apparent K+ affinity.
When K+ ions are absent during incubation, the enzymes are
presumably accumulated in the phosphorylated state. The hybrid ATPase NaKHK
was phosphorylated to 21% of the
Na+,K+-ATPase phosphorylation level, which is
slightly higher than the percentage activity obtained in the ATPase
reaction. This lower ATPase activity and phosphorylation level of the
hybrid must mainly be caused by a less efficient subunit assembly.
Surprisingly, with the hybrid HK
NaK
we were not able to measure
any specific phosphorylation. Although no K+ is added to
the reaction mixture it still contained about 5 µM K+ as determined by flame photometry. The decrease in
K0.5 directs the enzyme from the
E2-P into the E2
conformation, while the decrease in IC50 directs the enzyme
from the E1 into the E2
conformation. Both these processes inhibit accumulation of hybrid
HK
NaK
in the E2-P conformation and drive
the enzyme into the E2 conformation. It is
unlikely, however, that the low amount of K+ present
accounts for the total absence of any phosphorylated intermediate.
Another explanation for the absence of a phosphorylated intermediate in
the hybrid HKNaK
is an increased turnover number. The
H+,K+-ATPase
-subunit decreases the turnover
number of the hybrid NaK
HK
as compared with that of NaK
significantly. The assumption that the
Na+,K+-ATPase
-subunit increases the
turnover number of the hybrid HK
NaK
, as compared with HK
,
seems likely. This higher turnover number would then be responsible for
a lower phosphorylation level for HK
NaK
. If the increase in
turnover number is of the same magnitude as the 1.8-fold decrease in
the turnover number of the hybrid NaK
HK
as compared with
NaK
, then the maximal EP level of HK
NaK
is 0.3 pmol mg
1 protein, which is below the detection limit.
The suggestion of a relationship between the K+ affinity
and the turnover of the ATPases, which both are influenced by the -subunit, is tempting. The deocclusion step for
Na+,K+-ATPase is the rate-limiting step, while
for the H+,K+-ATPase the dephosphorylation step
(this is the occlusion of K+) is rate-limiting. This
rate-limiting step must be accelerated if the turnover number is
raised. The hybrid HK
NaK
then not only has an increased
K+ affinity but also a higher rate of K+
stimulated dephosphorylation as compared with HK
. Thus,
HK
NaK
occludes K+ faster and with a higher affinity
than HK
, which directly increases the turnover number. The
opposite is true for the other hybrid NaK
HK
, although in this
case the K+ occlusion becomes rate-limiting.
The findings reported here show that both
Na+,K+-ATPase and
H+,K+-ATPase require their own -subunits for
optimal activity. Probably the subunit assembly in the hybrid ATPases
is less efficient than in the wild type ATPases. When the
-subunits
are exchanged, the enzyme activity decreases and the apparent
K+ affinity of both hybrid ATPases is modified. The
Na+,K+-ATPase
-subunit gives the enzyme a
higher K+ affinity and probably a higher turnover number
than the H+,K+-ATPase
-subunit.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Barnett for providing SCH
28080 and Drs. M. Caplan, J. Forte, W. J. Ball Jr., and J. V. Møller for generously providing the various antibodies. We also thank
Drs. G. E. Shull and J. B. Lingrel for providing the rat
cDNA clones of the H+,K+-ATPase - and
-subunits and the rat and sheep cDNA clones of the
Na+,K+-ATPase
1- and
1-subunits, respectively.
![]() |
FOOTNOTES |
---|
* This work was supported by Netherlands Foundation for Scientific Research (NWO-ALW) Grant 805-05.041.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. Tel.: 31-24-3614260;
Fax: 31-24-3540525; E-mail: J.dePont{at}bioch.kun.nl.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
X+, Na+ or H+;
HK, H+,K+-ATPase
-subunit;
NaK
, Na+,K+-ATPase
-subunit;
NaK
, Na+,K+-ATPase;
NaK
HK
, the hybrid
consisting of the Na+,K+-ATPase
-subunit and
the H+,K+-ATPase
-subunit;
HK
, H+,K+-ATPase;
HK
NaK
, the hybrid
consisting of the H+,K+-ATPase
-subunit and
the Na+,K+-ATPase
-subunit;
Sf, Spodoptera frugiperda;
PAGE, polyacrylamide gel
electrophoresis;
C12E8, octaethylene glycol
monododecylether;
SCH 28080, 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|