From the
High-affinity ouabain binding to
Na
The Na
Because yeast do not have an endogenous
Na
In this study the role of Na
For
experiments with AMP-PNP, the tetra-lithium salt of AMP-PNP (Sigma) was
used. Preliminary experiments suggested that the solution was
contaminated with
ATP-dependent Binding of
[
As shown in A, incubation of
When the same measurements were made using
It is interesting to note that
The
In the results reported here, effects of
differences in
Complexes of both
Measurement of ATPase
activity by ouabain-sensitive phosphate release from ATP shows that the
maximum turnover of ATP by
Overall, the chimeric NH
Ouabain binding to yeast membranes containing the
indicated subunits was measured after 3 min under the indicated
conditions and after 60 min in a Mg
Values for
K
We thank Daun S. Putnam and Ema Hrouda for technical
support. We thank Ronald Hitzeman (Genentech) for providing the YEp1PT
yeast expression vector and yeast strain 30-4, Edward Benz (Yale) for
providing the rat
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/K
-ATPase (sodium- and
potassium-transport adenosine triphosphatase (EC 3.6.1.37)) requires
phosphorylation of the
subunit of the enzyme either by ATP or by
inorganic phosphate. For the native enzyme (
/
1), the
ATP-dependent reaction proceeds about 4-fold more slowly in the absence
of Na
than when saturating concentrations of
Na
are present. Hybrid pumps were formed from either
the
1 or the
3 subunit isoforms of
Na
/K
-ATPase and a chimeric
subunit containing the transmembrane segment of the
Na
/K
-ATPase
1 isoform and the
external domain of the gastric
H
/K
-ATPase
subunit
(
/NH
1 complexes). In the absence of Na
,
these complexes show a rate of ATP-dependent ouabain binding from
75-100% of the rate seen in the presence of Na
depending on buffer conditions. Nonhydrolyzable nucleotides or
treatment of ATP with apyrase abolishes ouabain binding, demonstrating
that ouabain binding to
/NH
1 complexes requires
phosphorylation of the protein. Buffer ions inhibit ouabain binding by
/NH
1 in the absence of Na
rather than
promote ouabain binding, indicating that they are not substituting for
sodium ions in the phosphorylation reaction. The pH dependence of
ATP-dependent ouabain binding in the presence or absence of
Na
is similar, suggesting that protons are probably
not substituting for Na
. Hybrid
/NH
1 pumps
also show slightly higher apparent affinities (2-3-fold) for ATP,
Na
, and ouabain; however, these are not sufficient to
account for the increase in ouabain binding in the absence of
Na
. In contrast to phosphoenzyme formation and ouabain
binding by
/NH
1 complexes in the absence of
Na
, ATPase activity, measured as release of phosphate
from ATP, requires Na
. These data suggest that the
transition from E
P to E
P
during the catalytic cycle does not occur when the sodium binding sites
are not occupied. Thus, the chimeric
subunit reduces or
eliminates the role of Na
in phosphoenzyme formation
from ATP, but Na
binding or release by the enzyme is
still required for ATP hydrolysis and release of phosphate.
/K
-ATPase
(
)
is a membrane-embedded enzyme complex that utilizes energy
derived from ATP hydrolysis to transport Na
and
K
ions across cell membranes. The active enzyme
complex consists of two dissimilar subunit proteins, an
subunit
of approximately 1000 amino acids, and a
subunit of approximately
300 amino acids. The
subunit of
Na
/K
-ATPase is structurally and
evolutionarily related to a large gene family of P-type ATPases which
transport a number of different cations in organisms ranging from
archaebacteria to mammals. Amino acids that participate in the binding
of cardiac glycosides by
Na
/K
-ATPase
(1, 2, 3, 4, 5, 6) are located in the binding site for substrate
ATP
(7, 8) , or whose chemical modification inactivates
cation occlusion
(9, 10) , have been mapped to the
subunit. The Na
/K
-ATPases and
H
/K
-ATPases form a closely related
subfamily of P-type ATPases that are unique in their requirement for a
subunit. The role of the
subunit in
Na
/K
-ATPase or
H
/K
-ATPase function is poorly
understood. The formation of a complex between the
and
subunits is essential for enzyme activity
(11, 12) , and
there is evidence that complex formation occurs before
and
subunits are transported from the endoplasmic reticulum to the plasma
membrane
(13, 14) . Recently, new isoforms of the
Na
/K
-ATPase
subunit (
2 or
AMOG
(15, 16) ) and the
subunit for
H
/K
-ATPase
(HK
(17, 18, 19) ) have been identified
which show only 25-35% sequence identity with the original
Na
/K
ATPase
subunit isoform
(
1). Heterologous expression of these new
isoforms in yeast
and Xenopus oocytes has confirmed that they are also capable
of assembling with Na
/K
-ATPase
subunits into active enzyme complexes. Complexes with the HK
isoform, however, show differences in complex stability
(20) and
require higher concentrations of K
to stimulate ATP
hydrolysis and ion transport
(21, 22) . These results,
together with results from chemical modification of the
H
/K
ATPase
(23) and
Na
/K
-ATPase
(24) , raise the
possibility that the
subunit participates in ion binding and
active ion transport.
/K
-ATPase, expression of mammalian
Na
/K
-ATPase in yeast provides the
opportunity to test the functional properties of specific combinations
of
and
isoforms of
Na
/K
-ATPase. In an initial study,
chimeric
subunits were formed by combining the intracellular and
transmembrane regions of the rat
Na
/K
-ATPase
1 subunit and the
extracellular region of the rat gastric
H
/K
-ATPase
subunit (NH
1)
or the intracellular and transmembrane regions of the rat gastric
H
/K
-ATPase
subunit and the
extracellular region of the rat
Na
/K
-ATPase
1 subunit
(HN
1)
(20) . Co-expression of these chimeric
subunits
with either the sheep
1 or rat
3 subunits of
Na
/K
-ATPase in yeast cells resulted
in the formation of functional hybrid
Na
/K
-ATPase complexes that bind
ouabain with high affinity (K
5-15
nM). Ouabain binds with high affinity to a phosphoenzyme
intermediate in the Na
/K
-ATPase
reaction cycle formed from either ATP or inorganic phosphate
(P
)
(25, 26) . High affinity ouabain binding,
therefore, reflects the formation of active pump complexes. Ouabain
binding to phosphoenzyme formed from Mg
and ATP is
stimulated by the presence of Na
, whereas ouabain
binding to phosphoenzyme formed from Mg
and P
is inhibited by either Na
or
K
(27, 28, 29) .
Mg
- and P
-dependent ouabain binding to
complexes formed from
and chimeric
subunits was also
inhibited by both K
(20) and
Na
.
(
)
Structural features
located in the external domain of the
subunit appear to be
responsible for differences in the apparent K
affinity
between Na
/K
-ATPase
subunits
and H
/K
-ATPase
, whereas
differences in the stability of
/
1 and
/HK
complexes appear to map to the cytoplasmic/transmembrane region of the
subunit
(20) .
in promoting phosphoenzyme formation and ouabain binding from ATP
for various
/
combinations has been studied. Complexes formed
with either
1 and
1 or the HK
subunit show a similar
dependence on Na
for phosphorylation and ouabain
binding. After a short (3 min) incubation, more ouabain is bound at low
(1-2 mM) concentrations of Na
by
3/HK
complexes than by
3/
1 complexes, suggesting
that the rate of phosphoenzyme formation is faster in
3/HK
complexes. In contrast, complexes of
isoforms combined with the
NH
1 chimera were able to efficiently form phosphoenzyme from ATP
and bind high levels of ouabain in the absence of Na
.
In this case, ouabain binding still required phosphoenzyme formation
and was largely independent of buffer ions and pH, suggesting that
other ions were not substituting for Na
. However,
although
/NH
1 complexes would bind ouabain in an
ATP-dependent reaction in the absence of Na
, overall
ATP hydrolysis did not occur at measurable rates for
/NH
1
complexes in the absence of Na
. Thus, although
phosphoenzyme may form under these conditions, the enzyme still
requires Na
to complete the reaction cycle. These
results show that the structure of the
subunit can influence both
the interaction of the enzyme with Na
and the kinetics
of Na
/K
-ATPase reactions.
Materials
The yeast strain 30-4 (MAT , trp1, ura3, Vn2,
GAL
) was obtained from R. Hitzeman (Genentech) and was
used for all heterologous expression studies.
[
H]Ouabain (specific activity, 23-25
Ci/mmol) was purchased from DuPont NEN. cDNAs encoding the rat
3
and rat
1 subunits were obtained from Edward Benz (Yale), the
sheep
1 cDNA was obtained from Jerry Lingrel (University of
Cincinnati), and the rat HK
cDNA was obtained from Robert Levenson
(Yale). Clones of the sheep
1 cDNA in the vector YEp1PT
(YEpNK
), the rat
3 cDNA in the vector YEp1PT (YEpR
3),
the rat
1 cDNA in the vector pG1T (pG1T-R
1), and the rat
H
/K
-ATPase
subunit in the
vector pG1T (pG1T-HK
) have been described
previously
(12, 21, 30) . Construction of
chimeric
subunits (NH
1 and HN
1) and cloning into the
expression plasmid pG1T (yielding pG1T-NH
1 and pG1T-HN
1,
respectively) have been described previously
(20) .
Methods
Standard yeast media were used throughout this
study
(31) . The yeast strain 30-4 was transformed with different
combinations of the subunit expression plasmids (YEpNK
or
YEpR
3) and
subunit expression plasmids (pG1T-R
1,
pG1T-HK
, pG1T-NH
1, or pG1T-HN
1) using the LiAc procedure
of Ito
(32) . Following selection on minimal media for
transformation with both plasmids, frozen glycerol stocks were made of
transformants and were stored at -80 °C. Transformed colonies
for the different
+
combinations were grown in
YNB-galactose medium using a mixture of supplements
(31) omitting tryptophan and uracil to maintain selection for
the expression plasmids. A microsomal membrane fraction was isolated as
described (21). Protein concentrations were assayed by the method of
Lowry
(33) . Mg
- and P
-dependent
[
H]Ouabain Binding-Assays for
[
H]ouabain binding were done in duplicate using
(final concentrations) 20 nM [
H]ouabain,
4 mM MgCl
, 4 mM Tris PO
, 50
mM Tris-HCl, pH 7.4, and between 0.25 and 1 mg of membrane
protein/assay. Assays were rocked at 37 °C for 1 h, chilled on
ice/H
O for 15 min, and pelleted in a microcentrifuge
(Eppendorf) for 15 min at 4 °C. The tubes were rinsed briefly with
0.5 ml of ice-cold buffer, and pellets were suspended with 1% SDS prior
to scintillation counting. Nonspecific binding was determined in
duplicate by the addition of 1 mM non-radioactive ouabain and
was subtracted from assay values. A mock-assay was performed without
[
H]ouabain, and the pellet was solubilized in 1% SDS and was assayed for protein recovery. Membranes from untransformed
yeast or yeast transformed with vectors alone showed
[
H]ouabain binding equal to the nonspecific
binding measured with the addition of 1 mM non-radioactive
ouabain, typically
20 fmol of [
H]ouabain/mg
of microsomal protein.
ATP-dependent Ouabain Binding
Microsomal membranes from
yeast expressing the desired /
combinations were washed free
of Na
by diluting the membranes about 25-fold in
either sodium-free buffer (25 mM Imidazole, 1 mM EDTA
(free acid), pH 7.4) or distilled water, and membranes were pelleted
for 60 min 4 °C at 50,000 rpm in a Beckman Ti-70 rotor. The
membrane pellet was suspended in sodium-free buffer or distilled water
and was pelleted again before being suspended in a small volume of
buffer or distilled water. Microsomal membranes and reaction tubes for
ATP-dependent ouabain binding were prewarmed for 10 min at 37 °C,
and the reaction was initiated by the addition of membranes. Reactions
were incubated for 3 min at 37 °C and were stopped by transfer to
ice/H
O. Bound [
H]ouabain was
separated from free [
H]ouabain by centrifugation
for 15 min, 4 °C in a microcentrifuge (Eppendorf), and aspiration
of the supernatant. The reaction tubes were rinsed with 0.5 ml of
ice-cold distilled water, and pellets were suspended in 200 µl of
1% SDS prior to scintillation counting. Final concentrations in the
total reaction volume were 20 nM
[
H]ouabain, 5 mM MgCl
, and
appropriate concentrations of buffer (typically 35 mM
imidazole, pH 7.4), ATP (Tris salt, typically 100 µM)
(Sigma), and NaCl as indicated. Mg
- and
P
-dependent ouabain binding was performed on washed
membranes at the same time as ATP-dependent ouabain binding in order to
estimate the total number of ouabain binding sites available and the
protein recovery during separation of bound and free
[
H]ouabain. Protein recovery was typically around
70%. Assays were done in duplicate and nonspecific binding, determined
by the addition of 1 mM unlabeled ouabain, was subtracted from
assay values. Stock solutions of 10
or greater concentrations
of all reagents and washed membranes were assayed by flame photometry
for Na
. In all cases, the reagents failed to register
a measurable amount of Na
, although the sensitivity of
the instrument was 1 mM Na
minimum.
0.5% ATP, so 1 ml of a 0.5 mM AMP-PNP
solution was treated with 50 units of apyrase (Sigma) for 30 min at 30
°C before use. A similar solution of ATP was also treated with
apyrase as a control for complete conversion of ATP to AMP.
Measurement of ATPase Activity by P
Yeast microsomal membranes were extracted with
SDS (34) as described previously
(20) . Membranes were washed
free of NaRelease
as described above and were assayed for
ATPase activity by phosphate release
(7, 8) . Sodium
concentrations were as indicated in the figures, and potassium was 10
mM in all assays. Each reaction contained 80 µg of yeast
membrane protein and was incubated at 37 °C for 30 min.
Data Analysis
For titrations with ATP, the
measured levels of [H]ouabain bound were plotted
against the increasing concentrations of ATP added. Data were fit by a
first order reaction model [Bound = Bound
[ATP]/(K
+
[ATP]) + C] using the nonlinear curve
fitting function (Levenberg-Marquardt algorithm) of Slidewrite Plus for
Windows (Advanced Graphics Software, Inc., Carlsbad, CA). The constant
term was added to the equation to account for the small amount of
ATP-independent binding that was observed in the reaction in the
absence of ATP. As shown in Fig. 2and 3, this binding could
largely be attributed to
Mg
+P
-dependent binding. For the
Na
dependence of ATP hydrolysis, measured levels of
P
released (expressed as percent of maximal activity) were
plotted against increasing concentrations of Na
. Data
were fit by the Hill equation for multisite reaction mechanisms [%
activity = 100
[Na]
/{[Na]
+
(K
)
}], using the
method described above to give estimates of the K
for Na
and the Hill coefficient
n
.
Figure 2:
ATP-dependent
[H]ouabain binding by
1/
1 and
1/NH
1. Microsomal membranes from yeast expressing the
indicated
/
subunits were washed free of Na
and assayed for ATP-dependent [
H]ouabain
binding in 35 mM imidazole/HCl, pH 7.4, 5 mM
MgCl
with or without 100 µM Tris-ATP and with
or without 10 mM NaCl as indicated in the figure. Nonspecific
binding in the presence of 1 mM ouabain was subtracted from
all values. Bars indicate the average of duplicate points and
the variation between duplicates, including variation in the
nonspecific binding. The percent specific binding for each condition,
normalized to 50 µM ATP, 10 mM Na
as 100%, is indicated under each
bar.
H]Ouabain-During the transport of
Na
and K
,
Na
/K
-ATPase undergoes a reaction
sequence that includes Na
binding at the intracellular
face of the enzyme, ATP hydrolysis, Na
transport
across the plasma membrane, K
binding at the
extracellular face of the enzyme, and its transport to the cytoplasm of
the cell (Fig. 1A)
(35, 36) . Ion
translocation is thought to occur during transitions between two major
conformations of the enzyme (E1 and E2). Additional
conformational intermediates have been inferred from the sensitivity of
the phosphoenzyme to ADP and
K
(26, 37) . The cardiac glycoside
ouabain binds with high affinity to
Na
/K
-ATPase when it is phosphorylated
and is in the E*P conformation
(26) . Little or no
ouabain binding is detectable in the absence of phosphorylation
indicating that the affinity of nonphosphorylated conformations of the
pump for ouabain is much lower than the E*P conformation.
Under normal physiological conditions, sodium binding to the enzyme
promotes the efficient formation of phosphoenzyme from ATP
(``front door'' phosphorylation), and subsequent ouabain
binding. Phosphoenzyme may also be formed by incubating the enzyme with
Mg
and P
(``back door''
phosphorylation; Fig. 1B). In this case, the addition of
Na
to the reaction will drive the enzyme toward the E1
conformation and will inhibit ouabain binding. Mg
is
a required co-factor in both the front door and back door
phosphorylation reactions. Since the hydrolysis of ATP leads to release
of P
, phosphoenzyme formation by either reaction is
possible under front door conditions.
Figure 1:
Schematic representation of the
reaction cycle of Na/K
-ATPase and the
interaction with ouabain. (A) reaction scheme for
ATP-dependent Na
and K
transport by
Na
/K
-ATPase. Adapted from Ref. 39,
with modifications based on Ref. 26. B, reaction scheme for
Mg
+ P
-dependent ouabain binding by
Na
/K
-ATPase.
Fig. 2
shows ouabain
binding to either 1/
1 (A) or
1/NH
1
(B) under front door phosphorylation conditions. A membrane
fraction from yeast cells expressing the indicated subunits was washed
free of Na
by a 25-fold dilution in
Na
-free buffer and sedimentation in an ultracentrifuge
twice before being resuspended in Na
-free buffer. The
sodium concentration in all reagents was below the detection limit of
flame photometry. Membrane samples were incubated in the presence of 20
nM [
H]ouabain, 5 mM
Mg
, and in the absence or presence of 100
µM Tris-ATP and/or 10 mM Na
as
indicated. The reaction was allowed to proceed for 3 min in order to
ensure that ATP was not depleted and that a significant level of
P
, which would lead to back door ouabain binding, was not
released. After the 3-min incubation, membranes were collected by
centrifugation.
1/
1
with ATP in the presence of Na
resulted in the highest
amount of ouabain bound in 3 min. The amount of ouabain bound in 3 min
under these conditions was 25% of the total ouabain binding capacity of
these membranes as determined in an equilibrium binding assay
(). In the absence of ATP and Na
, ouabain
binding to
1/
1 was only 5.3% of the amount bound in the
presence of ATP and Na
(1.3% of total binding),
indicating that ouabain binding in this assay required the presence of
ATP. The addition of Na
in the absence of ATP reduced
ouabain binding almost to zero. This indicates that, in the absence of
ATP, ouabain was binding was to phosphoenzyme formed from
P
, since Na
will compete in the back door
reaction for phosphoenzyme formation and ouabain binding. Measurements
of P
in yeast membranes incubated similarly without ATP
showed that
2 µM P
is present in the
samples, consistent with this suggestion.
(
)
This
phosphate is likely to be derived from the breakdown of membrane
phospholipids. Incubation of
1/
1 complexes for 3 min with ATP
in the absence of Na
resulted in 36% of the amount of
ouabain bound compared with ATP + Na
conditions.
While some of this can be accounted for by a small amount of back door
phosphorylation and ouabain binding (
5% of ATP +
Na
), there is clearly some phosphorylation and ouabain
binding that is ATP dependent even in the absence of
Na
. Since formation of the phosphoenzyme is the
rate-limiting step in ouabain binding
(38, 39) , this
indicates that Na
is not required for ATP-dependent
phosphorylation. Instead, for normal Na
/K
ATPase complexes of
1/
1, the formation of phosphoenzyme
from ATP in the absence of Na
proceeds more slowly
than in the presence of Na
. Control experiments show
that when dog kidney microsomes containing
1/
1 are used,
maximal binding is observed with ATP + Na
,
whereas in the absence of Na
about
35% of the
ouabain binding seen with ATP + Na
is observed
(data not shown). This indicates that changes caused by expression of
1/
1 in yeast such as differences in glycosylation or an
altered phospholipid content of the membranes are not responsible for
these observations.
1/NH
1 (Fig. 2B), maximum ouabain binding after
3 min was also observed in the presence of ATP and Na
.
In this instance, 33% of the total ouabain binding capacity was reached
after 3 min (). Like with
1/
1, a small amount of
ouabain was bound to
1/NH
1 in the absence of ATP (19.5% of
the amount bound in the presence of Na
and ATP; 6.5%
of total sites), and this was reduced in the presence of Na
(4.2% of ATP + Na
; 1.4% of total sites),
suggesting some back door phosphorylation of the pump. In contrast to
1/
1, however, incubation of
1/NH
1 with ATP in the
absence of Na
resulted in a relatively high level of
ouabain binding after 3 min (74% of ATP + Na
). If
ouabain binding by
1/NH
1 complexes is limited by the
formation of phosphoenzyme as it is for
1/
1 complexes, then
this result suggests that phosphorylation of
1/NH
1 from ATP
in the absence of Na
proceeds more rapidly than
phosphorylation of
1/
1 under the same conditions. As shown
below, similar results were also obtained for the
3 pump
complexes, indicating that the NH
1 subunit has a general effect of
increasing the rate of phosphoenzyme formation from ATP in the absence
of Na
.
Phosphorylation Is Required for ATP-dependent Ouabain Binding
to
The high level of ATP-dependent
ouabain binding to /NH
1 Complexes
/NH
1 complexes in the absence of
Na
might be explained if ATP hydrolysis and
phosphoenzyme formation were no longer required for high affinity
ouabain binding. This could occur, for example, if nucleotide binding
were sufficient for the hybrid pump to fold into a conformation that
resembles the phosphoenzyme. In the experiment shown in Fig. 3,
ouabain binding to
3/
1 or
3/NH
1 complexes was
measured after a 3-min incubation with either 0 or 10 mM added
Na
in the presence of either 50 µM ATP,
no ATP or 50 µM AMP-PNP. As shown in
Fig. 3A, ouabain binding to
3/
1 complexes was
highest in the presence of ATP and 10 mM Na
,
as previously observed for
1 complexes. Similar to the results
with
1/
1,
3/
1 complexes gave only a low level of
ouabain binding in the presence of ATP and the absence of Na
(13% of ATP + Na
). Deleting ATP from the
reaction resulted in only low levels of ouabain binding in either the
absence of Na
(9% of ATP + Na
)
or in the presence of 10 mM Na
(4% of ATP
+ Na
). Incubation of
3/
1 in the
presence of the nonhydrolyzable ATP analog AMP-PNP gave only low levels
of ouabain binding similar to those seen in the absence of ATP. If ATP
was hydrolyzed to AMP and P
by incubation with apyrase
before the reaction, a low level of ouabain binding was observed that
was identical to samples incubated with the same concentration of
P
for 3 min (data not shown). This confirms that ouabain
binding from ATP requires a phosphate that can be transferred from the
nucleotide to the enzyme.
Figure 3:
Phosphoenzyme formation is required for
front door ouabain binding. Front door ouabain binding to 3/
1
(A) or
3/NH
1 (B) was done for 3 min in the
presence of 50 µM Tris ATP (+ATP), without
ATP (-ATP), or 50 µM AMP-PNP
(AMP-PNP). Assays were done in duplicate either in the absence
of Na
or the presence of 10 mM NaCl as
indicated in the figure. Bars represent the mean of duplicate
values with error bars indicating the variation. The percent
specific binding for each condition, normalized to 50 µM
ATP, 10 mM Na
as 100%, is indicated under
each bar.
A similar dependence of ouabain binding on
ATP was observed for 3/NH
1 complexes
(Fig. 3B). The maximum amount of ouabain was bound after
3 min in the presence of ATP and Na
. However, a high
level of ouabain binding by
3/NH
1 (68% of ATP +
Na
) was also observed in the presence of ATP and the
absence of Na
, similar to the results in
Fig. 2
for
1/NH
1. In the absence of ATP, only
7-14% of the amount of ouabain bound in the presence of ATP and
Na
was observed. As with
3/
1, ouabain
binding in the absence of ATP was inhibited by the addition of
Na
, suggesting that it is due to back door
phosphorylation of the pump. Substitution of AMP-PNP for ATP resulted
in only low levels of ouabain bound by
3/NH
1, similar to
samples with no ATP added. Likewise, if ATP was hydrolyzed to AMP and
P
by incubation with apyrase before the assay, ouabain
binding was inhibited by the addition of 10 mM Na
in an identical fashion to samples incubated with the same
concentration of P
for 3 min (data not shown). This
indicates that nucleotide binding to
/NH
1 complexes alone is
not sufficient to support ouabain binding in this assay. These results
indicate that high-affinity ouabain binding by complexes formed between
Na
/K
-ATPase
subunits and either
the native
1 subunit or the chimeric NH
1 subunit requires the
hydrolysis of ATP and the formation of a phosphoenzyme intermediate.
/NH
1 complexes show more
ouabain binding over a short time course than
/
1 complexes.
This is seen with both
1 and
3 isoforms, and with both front
door and back door phosphorylation conditions. In , the
amounts of ouabain bound from either (ATP + Na
)
or P
in 3 min are compared with the total amount of ouabain
binding sites present in the same membranes. For
1/
1, front
door phosphorylation (ATP + Na
) results in 25% of
the total ouabain binding sites occupied in a 3-min assay, whereas in
the same conditions 33% of
1/NH
1 sites will bind ouabain.
Likewise, ouabain will bind to 40% of
3/
1 sites in 3 min from
ATP, whereas 45% of
3/NH
1 sites will be occupied in the same
time. When 100 µM P
is used to form
phosphoenzyme, a smaller fraction of sites are occupied in 3 min,
indicating that phosphorylation from P
is kinetically
slower than phosphorylation from ATP. However, complexes of
/NH
1 still show more ouabain binding from P
under
these conditions than complexes of
/
1. In the case of
1,
1/
1 complexes show 9% of total sites occupied in 3 min,
whereas
1/NH
1 complexes show 15%. Complexes of
3 show a
similar increase from 9 (
3/
1) to 22% (
3/NH
1) of
total sites occupied. Although the effect is small in magnitude, our
experiments consistently show that the presence of the NH
1 subunit
increases the amount of ouabain binding seen in a short time course.
Since the formation of the phosphoenzyme is the rate-limiting step in
ouabain binding, this suggests that the NH
1 subunit slightly
increases the rate of phosphoenzyme formation from either ATP or
P
.
The Role of Buffer Ions in
Na
Certain buffer ions have been reported to
have Na-independent Ouabain Binding by
/NH
1
-like effects on the conformation of
Na
/K
-ATPase
(40, 41) .
Schuurmans Stekhoven et al.(40, 41) have
reported that at low concentrations of Mg
(0.1
mM), imidazolium ion will substitute for Na
in promoting the formation of phosphoenzyme from ATP by
Na
/K
-ATPase. This effect is specific
to low concentrations of Mg
and is nearly eliminated
at 2.5 mM Mg
. Substitution of
Tris
for imidazolium ion does not support
ATP-dependent phosphorylation of the pump. Because the effect of
imidazole is increased at higher concentrations and is specific for the
imidazolium ion, it was suggested that imidazolium ion may substitute
for Na
in the phosphorylation reaction.
Fig. 4
shows the results of an experiment to test whether the
substitution of buffer ions for Na
ions could explain
the increased binding of ouabain to
/NH
1 complexes in the
absence of sodium. In this experiment, membranes were washed by
centrifugation in distilled water to remove Na
and
buffer ions. For
3/
1 (A), less than 30% of maximal
ouabain binding was observed after 3 min in the absence of
Na
and in the presence of either 5, 20, or 50
mM Tris or imidazole. A similar result was observed (40%
maximal binding) if buffer ions were excluded from the reaction, except
for a small amount of Tris
from the 100
µM Tris-ATP solution used. For
3/NH
1
(B) the same amount of ATP-dependent ouabain binding was
observed in unbuffered water in the presence or absence of 10
mM Na
. Buffer ions do have some effects on
the reaction, with higher amounts of ouabain bound in the presence of
Na
at low buffer concentrations compared with water
and less ouabain bound in the absence of Na
. Compared
with
3/
1, however, the
3/NH
1 complex still binds
2-3-fold more ouabain in the absence of Na
under
all buffer conditions tested. Although a small amount of
Tris
could not be eliminated from the reaction,
increasing the concentration of Tris
inhibits ouabain
binding in the absence or presence of Na
. Thus, it
does not appear that the increased level of ATP-dependent ouabain
binding by
/NH
1 complexes in the absence of Na
is due to substitution of buffer ions for Na
.
Figure 4:
The effects of buffer ions on
ATP-dependent [H]ouabain binding. Microsomal
membranes from yeast expressing the indicated
/
subunits were
washed free of Na
using distilled water and were
assayed for front door [
H]ouabain binding in
either the absence of buffer (H
O) or in the buffer
indicated (pH 7.4) at concentrations of either 5, 20, or 50
mM, with 100 µM Tris-ATP and with 10 mM
NaCl (solid bars) or without Na
(open
bars) as indicated in the figure. A shows binding to
3/
1 complexes, and B shows binding to
3/NH
1 complexes. Variation between duplicates was <10 fmol
of ouabain bound in all cases (usually <2 fmol). The percent numbers
on the open bars indicate the fraction of binding in the absence of
Na
relative to the binding observed for the same
buffer conditions in the presence of 10 mM
Na
.
Na
In the experiment shown in Fig. 5,
the NaTitration of ATP-dependent
Ouabain Binding
requirement for ATP-dependent ouabain binding
by Na
/K
-ATPase complexes of
1,
3, and the
1, HK
, NH
1, and HN
1 subunits was
determined. The combination
3/HN
1 appears to either assemble
poorly or form an unstable complex when expressed in yeast and gives
levels of ouabain binding which are too low to test
reliably
(20) . As shown in Fig. 5, the amount of ouabain
bound by the combinations
1/
1,
1/HK
, and
3/
1 increases as [Na
] is
increased, with half-maximum ouabain bound at 1-2 mM
Na
for
1/
1 or
1/HK
or at 3-4
mM for
3/
1. For
3/HK
, a 2-fold increase in
the amount of ouabain binding is observed in the range of 1-2
mM Na
, compared with
3/
1. In all
four cases, approximately 20-30% of maximal ouabain binding is
observed in the absence of Na
, again suggesting that
the ATP-dependent phosphorylation and ouabain binding can proceed to a
limited extent in the absence of Na
. For
1/NH
1 and
3/NH
1, 65-75% of maximum ouabain
binding is observed in the absence of Na
. Addition of
Na
increases the amount of ouabain bound, with a
K
of approximately 0.6-0.8 mM.
These results indicate that the
/NH
1 complexes have only a
slightly higher apparent affinity for Na
than
/
1 complexes. One possible explanation for high levels of
ATP-dependent ouabain binding by
/NH
1 in the absence of
Na
would be if the
/NH
1 complexes have a
high affinity for Na
such that trace levels of
contaminating Na
in the reaction would be sufficient
to promote phosphorylation and ouabain binding. However, addition of
50-200 µM Na
to the reaction
results in little or no increase in ouabain binding consistent with the
absence of a very high affinity Na
site on the
/NH
1 complexes.
Figure 5:
[Na] dependence
of ATP-dependent ouabain binding. Microsomal membranes were washed with
Na
-free imidazole buffer and assayed in the front door
ouabain binding assay with increasing concentrations of NaCl. A shows the
1 subunit isoform combined with different
subunits. B shows the
3 subunit isoform combined with
different
subunits. Duplicate points for each concentration of
Na
are shown and lines connect the mean values at each
concentration. The leftmost point in each assay shows binding in the
absence of added Na
. Nonspecific binding was
subtracted from each data set, and data are expressed as the percent
maximum for each
/
combination.
Na titration of
ATP-dependent ouabain binding by
1/HN
1 complexes shows a
behavior complementary to that of the
1/NH
1 complexes. Here,
ouabain binding in the absence of Na
is less than the
1/
1 complexes, and the K
for
Na
has increased to approximately 10 mM.
Thus, although the NH
1 chimera seems to decrease the Na
requirement for phosphoenzyme formation from ATP and increase the
apparent Na
affinity, the complementary
subunit
chimera HN
1 seems to increase the Na
requirement
for phosphoenzyme formation from ATP and decrease the apparent
Na
affinity.
pH Dependence of Front Door Ouabain
Binding
Protons have been reported to serve as substitutes for
Na ions in
Na
/K
-ATPase
reactions
(42, 43) . In order to examine whether protons
are substituting for sodium ions in the ATP-dependent binding of
ouabain to
/NH
1 complexes, the pH dependence of this reaction
was examined (Fig. 6). The amount of ouabain bound by both
3/NH
1 complexes and
3/
1 complexes decreased as the
pH was increased from 6.8 to 8.0 in the presence of 10 mM
Na
. In the absence of Na
, ouabain
binding by
3/NH
1 complexes decreased from 71% of control in
the presence of 10 mM Na
at pH 6.8 to 55% of
control at pH 8.0. The
3/
1 complexes also show a small
decrease in fractional ouabain binding from 32 to 26% as pH was
increased from pH 6.8 to 8.0. The reduction in ATP-dependent ouabain
binding as pH was increased is consistent with the substitution of
protons for Na
in the phosphorylation reaction,
however, the magnitude of this reduction binding is small compared with
the change in pH which represents more than an order of magnitude
change in [H
] from pH 6.8 to pH 8.0.
Moreover, the pH-dependent change in ouabain binding in the presence of
10 mM Na
is at least as great as the the
changes observed in the absence of Na
. The pH
dependence of ouabain binding is similar to the pH dependence of
phosphoenzyme formation by
Na
/K
-ATPase reported by Forbush and
Klodos
(44) . This suggests that protons are playing a similar
role for both the normal
/
1 complexes and
/NH
1
complexes. Nevertheless, it is hard to rule out a role for H
in this reaction entirely since a change in pH can also affect
the ionization state of charged amino acid side chains that may
participate in cation binding and/or transport.
Figure 6:
Effects of pH on ATP-dependent ouabain
binding. Microsomal membranes with 3/
1 (left panel)
or
3/NH
1 complexes (right panel) were washed with
distilled water and were assayed in the front door ouabain binding
assay using 25 mM imidazole buffer at the pH values indicated
in the figure. Solid bars indicate binding done in the
presence of 10 mM NaCl, and open bars indicate
binding in the absence of added Na
. Variation between
duplicates was <20 fmol [
H]ouabain bound in
all cases (usually <5 fmol). The percentage on the open bars indicates the fraction of binding in the absence of Na
relative to the binding observed for the same buffer conditions
in the presence of 10 mM
Na
.
The Effect of the NH
Scatchard analysis of equilibrium
[1 Subunit on Ouabain
Affinity
H]ouabain binding to both
3/
1 and
3/NH
1 complexes indicates that the pumps assembled with the
chimeric
subunit had about a 2-fold higher affinity for ouabain
than the native pumps (data not shown). The K
for ouabain binding to
3/
1 complexes was 15.2 ±
4.1 nM (n = 4; ± S.D.), whereas the
K
for ouabain binding to
3/NH
1
complexes was 6.7 ± 1.4 nM (n = 3;
± S.D.). A similar increase in affinity for ouabain has
previously been observed when
Na
/K
-ATPase
subunits are
assembled with the gastric H
/K
-ATPase
subunit
(21) .
Affinity of Pump Complexes for ATP
In
Fig. 7
, the apparent affinity of both 1/NH
1 complexes
and
1/
1 complexes for ATP was estimated by measuring the ATP
concentration dependence of ouabain binding in the absence and presence
of Na
. For
1/
1 complexes, the total amount
of ouabain binding increases about 4-fold in the presence of added
sodium, whereas for
1/NH
1 complexes there is only a slight
increase in total binding, similar to previous results. The apparent
affinity (K
) of
1/
1 complexes for ATP
in the absence of Na
was 1.8 µM, and this
increased to 0.8 µM with the addition of 10 mM
Na
. The apparent affinity of
1/NH
1 complexes
for ATP in the absence of Na
was 1.3 µM,
and this increased to 0.3 µM with the addition of 10
mM Na
. Thus, under equivalent conditions,
1/NH
1 complexes have a slightly higher apparent affinity for
ATP than
1/
1 complexes. The concentrations of ATP used in the
experiments described in this report (50-100 µM ATP)
are sufficient to saturate ATP binding both in the presence and absence
of Na
for both
/
1 and
/NH
1
complexes. Thus, although
/NH
1 complexes have a higher
affinity for ATP, this increase in affinity is not sufficient to
account for the increased level of ouabain binding observed in the
absence of Na
. The differences in affinity for ATP
between
/
1 and
/NH
1 complexes are another example
of the influence of
subunit structure on enzyme properties.
Figure 7:
Titration of ATP requirement for front
door ouabain binding. Microsomal membranes with 1/
1
(A) or
1/NH
1 complexes (B) were washed with
Na
-free buffer and assayed in the front door ouabain
binding assay with increasing concentrations of Tris-ATP. The
+ symbols indicate titrations done in the absence of
added Na
and the
symbols indicate
titrations done in the presence of 10 mM NaCl. Curves drawn
show the fit to the data of a single site reaction model with a
constant term added to account for ATP-independent ouabain binding in
the reaction. The calculated constant values subtracted were 10.4 fmol
(
1/
1, 0 Na
), 13.2 fmol (
1/
1, 10
Na
), 22.8 fmol (
1/NH
1, 0
Na
), and 4.4 fmol (
1/NH
1, 10
Na
). Correlation coefficients (r
)
for the data fits were 0.99 for
1/
1, at 0 and 10
Na
, and for
1/NH
1 at 10 Na
,
and 0.96 for
1/NH
1 at 0 Na
. The apparent
affinities for ATP in the reactions are indicated in the
figure.
ATP Hydrolysis by
The high level of phosphoenzyme formation and ouabain
binding by /NH
1 Complexes Requires
Sodium
/NH
1 complexes in the absence of Na
suggests that hydrolysis of ATP by these complexes may no longer
be Na
-dependent and raises the possibility that ATP
hydrolysis is not coupled to Na
transport in these
complexes. To examine this, the Na
concentration
dependence of ATP hydrolysis by different pump complexes was determined
in an assay that measures release of P
. In Fig. 8,
the ATPase activities of
1/
1 and
1/NH
1 complexes
(A) or
3/
1 and
3/NH
1 complexes
(B) are plotted as a function of Na
concentration. For all of the
/
combinations, there was
no measurable ATPase activity in the absence of Na
.
Thus, although
/NH
1 complexes are able to form phosphoenzyme
and bind ouabain in the absence of Na
, they are unable
to proceed through the reaction cycle and release phosphate at
measurable rates in the absence of Na
. The data for
each
/
combination were normalized to the total number of
Na
/K
-ATPase complexes determined by
equilibrium ouabain binding in order to calculate the turnover number
for each complex, and the Na
concentration dependence
was fit by the Hill equation in order to obtain the apparent affinities
of each complex for Na
and the Hill coefficients for
the reaction (). For
1/
1 and
3/
1
complexes, the maximum turnover rate was
8000 ATP/min. Complexes
of
1/NH
1 and
3/NH
1 were also able to hydrolyze ATP
at maximum rates between about 6000 and 14,000 ATP/min. This indicates
that under optimal conditions,
/NH
1 complexes are efficient
ATPases and that the overall ability of the enzyme to proceed through
the reaction cycle has not been severely compromised by the structural
change in the
subunit. The apparent affinity
(K
) of
1/
1 and
3/
1 complexes
for Na
in this reaction is 9.3 and 14.8 mM,
respectively (). These values are higher than
K
values reported by Jewell and Lingrel
(45) for modified rat
1 (1.2 mM) and
3 (3.1
mM) isoforms expressed in HeLa cells, although the higher
apparent affinity of
1 than
3 for Na
that
was reported there is also seen here. The reasons for the quantitative
differences in K
are not known. Both
1/NH
1 and
3/NH
1 show an approximately 3-fold
increase in apparent affinity for Na
, when compared
with
1/
1 and
3/
1 complexes, with K
values of 3.6 and 4.4 mM, respectively. The apparent
affinity for Na
measured in this reaction reflects the
interaction of Na
with the enzyme at multiple reaction
steps, and it is not possible using these data to determine which
Na
-dependent step(s) in the reaction cycle have been
affected.
Figure 8:
Na dependence of ATP
hydrolysis. Yeast microsomal membranes containing either
1/
1,
1/NH
1,
3/
1, or
3/NH
1 pump complexes were
washed by centrifugation to remove Na
. Activity
measurements were carried out at 37 °C for 30 min in the presence
of 10 mM KCl, 5 mM MgCl
, 3 mM
Tris-ATP, 15 mM Tris-azide, 1 mM Na
EDTA,
and 50 mM Tris/HCl, pH 7.4. The concentration of NaCl was
varied between 0 and 100 mM as indicated. Data for 50
mM NaCl are not shown. Each data point represents at least
three experimental values obtained in duplicate, and the average values
(±S.E.) are presented as the percentage of maximum
Na
/K
-ATPase activity calculated after
subtraction of ATPase activity measured in the presence of 1
mM ouabain. Curves are drawn to show the fit to the data by a
highly cooperative Hill equation: [% maximum activity = 100
[Na
]/{[Na
]
+ (K
)}]. A,
1/
1 (
) and
1/NH
1 (▾); B,
3/
1 (
) and
3/NH
1 (
). Kinetic constants
for each complex are shown in Table II.
Complexes of either 1/
1 or
3/
1 have
Hill coefficients for the sodium dependence of ATP hydrolysis of 2.0
and 2.3, respectively (). When the NH
1 subunit is
substituted for
1, the
1/NH
1 and
3/NH
1
complexes show a decrease in the Hill coefficients to 1.0 and 1.3,
respectively. This decrease suggests either that the stoichiometry of
Na
binding and transport has been altered by the
NH
1 subunit or that the extent of cooperativity of Na
binding has been altered in these complexes.
subunit of
Na
/K
-ATPase shows extensive homology
to the larger family of P-type cation transport ATPases, and numerous
studies have identified sites within the
subunit that are
involved in ATP binding and hydrolysis. The role of the
subunit
as the catalytic subunit of the enzyme is clear. The role of the
subunit, which is unique to the subfamily of
Na
/K
-ATPase isoforms and the
H
/K
-ATPase is poorly understood.
Formation of a complex between the
and
subunits is required
for Na
/K
-ATPase to exhibit
Na
- and K
-stimulated ATP hydrolysis,
high affinity ouabain binding, and cation transport
activities
(11, 12) . Assembly of complexes between
Na
/K
-ATPase
subunit isoforms
and different isoforms of the
Na
/K
-ATPase
subunit or a
subunit from the gastric H
/K
-ATPase
in heterologous expression systems has made it possible to examine the
effect of different
subunit structures on
Na
/K
-ATPase activity and to infer
whether the
subunit may contribute to enzymatic activity. When
the H
/K
-ATPase
subunit is
substituted for the Na
/K
-ATPase
1 isoform, hybrid pumps are formed which show high affinity
ouabain binding and Na
and K
transport
(21, 22, 46) . Hybrid pumps of
Na
/K
-ATPase
1 or
3 subunits
and the H
/K
-ATPase
subunit are
more sensitive to extraction by SDS than
1/
1 or
3/
1
complexes
(20) , suggesting that they form less stable complexes.
In addition,
1/HK
and
3/HK
complexes require higher
concentrations of K
to inhibit ouabain binding or to
stimulate ATPase activity, suggesting that the HK
subunit lowers
the affinity of the pump complexes for
K
(20, 21, 22) . A similar
effect on K
affinity has been reported for the
3
isoform from Bufo marinus(47) . Chimeric polypeptides
between the
1 and HK
isoforms indicate that the stability of
/
complexes depends at least in part on the transmembrane
region of the
subunit, since
subunits containing the
transmembrane region of the HK
subunit either form less stable
complexes or do not assemble with
subunits
(20) . For
K
affinity, the results are less clear. In one case, a
chimera with the external domain of the HK
subunit expressed in
yeast seems to show the same effect on K
affinity as
seen with the whole HK
subunit
(20) , whereas a similar
chimera expressed in Xenopus oocytes shows behavior
intermediate between the
1 and HK
controls
(22) . These
differences may be due to the fact that
and
isoforms from
different species were used or to difficulties in distinguishing
between endogenous Na
/K
-ATPase
activity and the activity of heterologously expressed subunits in
Xenopus oocytes.
subunit structure on the interaction of
Na
/K
-ATPase complexes with
Na
have been examined. For complexes of normal sodium
pump isoforms (
1/
1 and
3/
1), Na
promotes the formation of a phosphoenzyme from ATP leading to
high affinity ouabain binding. Low levels of ATP-dependent
phosphorylation and ouabain binding occur in these complexes in the
absence of Na
( Fig. 2and Fig. 3). This
indicates that Na
, while not absolutely required for
ATP hydrolysis and phosphorylation, accelerates these reactions. An
increase in the rate of ATP-dependent phosphoenzyme formation by
Na
has been also been reported for
Na
/K
-ATPase in Torpedo electric organ
(29) . It is possible that differences in the
makeup of membrane lipids in yeast membranes contribute to this effect;
however, control experiments with dog kidney microsomes show a slow
rate of ATP dependent ouabain binding in the absence of Na
which is accelerated when 10 mM Na
is
added (data not shown). These studies indicate that this observation is
not unique to Na
/K
-ATPase expressed
in yeast cells. The substitution of the HK
subunit for
1 has
no effect on the Na
dependence of ouabain binding when
combined with the
1 subunit; however, when HK
is combined
with the
3 subunit, this complex shows an increased level of
ouabain binding in the presence of 1-2 mM Na
(Fig. 5). Since phosphoenzyme formation is rate-limiting
for ouabain binding
(38, 39) , it is likely that the
increased level of ouabain binding by
3/HK
at low
[Na
] is due to an increase in the rate of
phosphoenzyme formation.
1 or
3 and the
chimeric
subunit NH
1 show a dramatic increase in the level
of ATP-dependent ouabain binding after 3 min in the absence of
Na
, exhibiting from 70-100% of maximum binding
depending on buffer conditions. (Figs. 2-5). Phosphorylation from
ATP is still required by
/NH
1 complexes for high-affinity
ouabain binding, since AMP-PNP will not support front door ouabain
binding (Fig. 3). Buffer ions such as Tris
or
imidazolium inhibit the reaction in the absence of Na
,
making it unlikely that they are substituting for Na
.
Changing the pH of the reaction from 6.8 to 8.0 results in a small
decrease in the relative amount of binding in the absence of
Na
; however, this decrease is similar to the
pH-dependent decrease in the presence of 10 mM
Na
. These results suggest that the differences
observed are due to more general effects of pH on the reaction rather
than the substitution of H
for Na
.
The most likely explanation for the high levels of ouabain binding by
/NH
1 complexes in the absence of Na
is the
rapid formation of a phosphoenzyme under these conditions. In the
absence of Na
,
/
1 complexes are also able to
form a phosphoenzyme from ATP and bind ouabain, and Na
accelerates this reaction. It is possible that the
/NH
1
complexes adopt a conformation in the absence of Na
similar to the conformation of
/
1 complexes when
Na
is bound and that the phosphorylation reaction
proceeds by a similar mechanism in both cases. It also appears that
phosphoenzyme formation is faster for
/NH
1 complexes not only
from ATP, but also from P
(). The increased
rate of phosphorylation combined with the reduced role of Na
in phosphoenzyme formation by
/NH
1 complexes raises the
possibility that there may be subtle changes in the mechanism of ATP
hydrolysis and phosphoenzyme formation.
/NH
1 complexes is comparable with
/
1 complexes. While there is
3-fold increase in the
apparent Na
affinity of
/NH
1 complexes
compared with
/
1, Na
is still required by
the
/NH
1 complexes for measurable levels of ATP hydrolysis.
The similar rates of maximum ATP turnover for
/NH
1 and
/
1 complexes indicate that the
/NH
1 complexes are
efficient and functional ATPases and that overall enzyme activity has
not been severely compromised by the structural change to the
subunit. A 3-fold increase in apparent Na
affinity for
/NH
1 in this reaction is similar to the increases in
K
values estimated for the Na
dependence of ouabain binding for these complexes (Fig. 5).
Finally, since Na
is still required by
/NH
1
complexes for measurable P
release from ATP, despite the
ability of these complexes to form a phosphoenzyme from ATP in the
absence of Na
, it must be concluded that Na
is also required at some step in the reaction cycle other than
phosphoenzyme formation. The most likely step is in the transition
E1P
E2P which is thought to accompany release
of Na
at the extracellular surface of the cell
membrane (Fig. 1). Gadsby et al.(48) and
Hilgemann
(49) have shown that release of Na
is
voltage-sensitive. The data reported here suggest that both
/
1 and
/NH
1 are capable of moving through the
reaction cycle to the step where Na
would be released,
but that the reaction cycle cannot proceed beyond this step when the
ion sites are not occupied. In the presence of sodium, the
stoichiometry of ion transport by
Na
/K
-ATPase is 3 Na
ions transported per ATP molecule hydrolyzed. It is possible that
the structural differences between
1, HK
, and the chimeric
NH
1 subunit that result in changes in the apparent affinity for
Na
are the result of structural perturbations of only
one ion binding site or else the cooperative interactions between the
binding of multiple Na
ions to one or more sites could
be affected.
1 subunit appears to
reduce or eliminate the role of Na
in formation of a
phosphoenzyme from ATP. It is interesting to note that this effect of
the NH
1 subunit is not seen with either the
1 or the HK
subunits, which are the parent molecules for NH
1. Some effect is
seen with
3/HK
complexes in promoting ATP-dependent ouabain
binding at low (1-2 mM) concentrations of Na
(Fig. 5). These complexes show only a small amount of
ouabain binding in the absence of Na
, however,
suggesting that Na
is still important in increasing
the rate of phosphoenzyme formation. Thus, the influence of
subunit structure on Na
interactions with the pump can
not be attributed to one structural region of the
subunit, in
contrast to the effect of
subunit structure on interactions with
K
(21) . Instead, the results reported here
suggest that multiple regions of the
subunit interact with
multiple regions of the
subunit. With the chimeric NH
1
subunit, these interactions cause a reduction in the activation energy
barrier to phosphoenzyme formation from ATP that is normally reduced by
Na
. These results provide additional evidence that the
subunit is an intimate partner with the
subunit in the
formation and activity of
Na
/K
-ATPase.
Table:
Percentage of ouabain binding sites
occupied in 3 min
- and
P
-dependent reaction as described under ``Experimental
Procedures.'' All reactions were done in the presence of 5 mM MgCl
.
Table:
Sodium-dependent ATP hydrolysis by
yeast membranes expressing 1 and
3 subunits with
1 or
NH
1 subunits
(Na
), the maximum velocity of
ATP hydrolysis (µmol/min/mg), and the Hill coefficient
(n
) were obtained from the experiments shown in
Fig. 8. The turnover number was obtained from the ratio
V
/B
, where
B
is the number of ouabain binding sites
determined by equilibrium ouabain binding. Values represent the mean of
at least three different experiments done in duplicate.
/K
-ATPase, sodium- and
potassium-transport adenosine triphosphatase (EC 3.6.1.37);
1, the
sheep
1 subunit isoform of
Na
/K
-ATPase;
3, the rat
3
subunit isoform of Na
/K
-ATPase;
1, the rat
1 subunit isoform of
Na
/K
-ATPase; HK
, the
subunit of rat gastric H
/K
-ATPase;
AMP-PNP, adenosine 5`-(
,
)-iminodiphosphate.
3 and rat
1 cDNAs, Robert Levenson (Yale)
for providing the rat HK
cDNA and J. J. H. H. M. de Pont for
helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.