(Received for publication, January 16, 1996; and in revised form, March 18, 1996)
From the
The overall reaction of well-defined solubilized protomers of
Na,K-ATPase (one plus one
subunit) retains the dual ATP
dependence observed with the membrane-bound enzyme, with distinctive
ATP effects in the submicromolar and submillimolar ranges (Ward, D. G.,
and Cavieres, J. D.(1993) Proc. Natl. Acad. Sci. U. S. A. 90,
5332-5336). We have now found that the
K
-phosphatase activity of the
protomers is
still inhibited by 2`(3`)-O-(2,4,6-trinitrophenyl)adenosine
5`-diphosphate (TNP-ADP). What is most significant is that the TNP-ADP
effect can be observed clearly with protomeric enzyme whose high
affinity ATP site has been blocked covalently with fluorescein
isothiocyanate. We conclude that nucleotides can bind at two discrete
sites in each protomeric unit of Na,K-ATPase.
The sodium pump or Na,K-ATPase ()uses the energy of
ATP hydrolysis to power the active transport of Na
and
K
ions across the plasma membrane(1) . This
integral membrane enzyme consists of
and
subunits. The
chain presents an ATP-binding site, a phosphorylation site, and a
ouabain-binding site. The
subunit is a glycoprotein of unclear
function, found in equimolar proportions with the
chain. We
recently reported (2) that solubilized
protomers of
highly purified sodium pump responded to ATP with high and low affinity
effects, and also that the complex behavior did not result from
protomer association to form soluble (
)
dimers.
Those experiments demonstrated that a complex dependence on nucleotides
was intrinsic to the
protomer. They could not decide,
however, whether this arose from the presence of more than one ATP site
per
protomer or from a single site whose function and
affinity changed round the catalytic cycle. That is the question
addressed in this paper.
It has long been recognized that ATP has
two types of activatory effect during the sodium pump
cycle(3) . Na,K-ATPase becomes phosphorylated by ATP during the
course of the reaction, and the small high affinity activation (K < 1 µM) correlates well
with the ATP requirement for phosphorylation of the E
form
of the enzyme(4) . The considerable low affinity stimulation of
the overall cycle (K
approximately
150-300 µM) seems to result from accelerating a
rate-limiting step in the E
form of the
dephosphoenzyme(4, 5) . ADP(6) ,
nonphosphorylating ATP analogues(7) , and acyl coenzymes A (8) can replace ATP in this low affinity effect. For these
reasons, this is regarded as a regulatory nucleotide effect, in
contrast with the ``catalytic'' (high affinity) ATP action
that results in enzyme phosphorylation in the presence of Na
ions. Affinity probes have so far returned what seems to amount
to a single, high affinity ATP site on the
chain. The purine ring
subsite has been mapped with 2-azido-ATP and
8-azido-ATP(9, 10) , which label peptides identifying
Gly
and Lys
, respectively, as the anchoring
points. This is a region which had earlier been found to be labeled by
FITC, after binding covalently to
Lys
(11, 12) . Probes for the
5`-triphosphate moiety of ATP, on the other hand, have identified a
sequence between Asp
and Lys
on the
chain(13, 14) .
The structural findings have
polarized the rationalization of the two ATP actions toward two basic
models: (i) a unique ATP-binding site whose affinity and function
change round the reaction cycle and (ii) a membrane ()
dimer, with one ATP site per
subunit, where the interaction
of
units leads to a degree of half-of-the-sites reactivity (15) during turnover. Evidence for the first arises from
studies with the vanadate-inhibited enzyme(16) , from
experiments with TNP-ATP, a high-affinity ATP analogue(17) ,
and, at least superficially, from the enzymatic competence of the
protomer in solution(2) . There are a number of
observations, however, that cannot be explained by a single
site(18) , and it now seems increasingly likely that TNP-ADP
can access more than one class of binding sites(19) .
A
third possibility, i.e. the idea of two distinct nucleotide
sites per unit, has not had much currency because of the
lack of supporting structural data, the controversy above, and a
certain sense of economy. This was, however, a viable alternative
hypothesis in the case of the soluble
protomer(2) .
We have approached the question by examining the behavior of soluble
protomers whose high affinity ATP site has been irreversibly blocked by
FITC. The covalently bound fluorescein suppresses phosphorylation by
ATP and obliterates the overall pump cycle, but does not affect
backwards phosphorylation by inorganic phosphate, or the
K
-phosphatase activity of the
enzyme(19, 20, 21) . A realistic setting was
that the K
-phosphatase substrate binds at a place
other than the FITC-blocked site.
The K-phosphatase
activity is the ability of the sodium pump to hydrolyze substrates like pNPP, carbamyl phosphate, and 3-OMFP, hydrolyses which do not
support cation fluxes. It is an E
-type of activity on
account of not only its K
requirement but the effect
of nucleotides on the reaction also. In fact, in the absence of
Na
ions, the K
-phosphatase activity
is inhibited, with low affinity, by ATP and other
nucleotides(22, 23) , including analogues like TNP-ADP (19) . We have used this effect to probe for a low affinity
nucleotide site in FITC-modified, soluble
protomers and
their parallel FITC-free controls. We found that TNP-ADP had a clear
inhibitory effect, as would only be expected if one high affinity and
one low affinity nucleotide site co-existed on the same protomeric
unit. A preliminary report has been published(24) .
Figure 1:
Inactivation of membrane-bound sodium
pump by FITC: two experiments. The enzyme (2.4 mg of protein/ml) was
incubated with 20 µM FITC for the times shown. Open
symbols, ATP protection. Na,K-ATPase activity remaining after FITC
treatment in the absence (open squares) and the presence (open circles) of 3 mM ATP. Solid symbols,
an experiment, under similar inactivation conditions, to measure the
K-phosphatase activity (solid circles) as
well as ATP hydrolysis (solid squares). The substrate
concentrations were 1 mM [
-
P]ATP
for Na,K-ATPase assays and 40 mMpNPP (Tris) for the
K
-phosphatase assays.
Our results for the inactivation of the Na,K-ATPase activity
of the sodium pump by FITC, the protective effect of
ATP(20, 29) , and the survival of the
K-phosphatase activity (20, 21) are
shown in Fig. 1, for illustration purposes. We could also
confirm that the K
for pNPP increases
(approximately 4-fold in our hands) following irreversible FITC
binding. The maximal K
-phosphatase rate remained high,
at about 80% of the control activity (see Table 2and legend to Fig. 4). In the experiments of Fig. 2and Fig. 3,
therefore, the K
-phosphatase activities used in the
calculations were the fitted V
values (estimated
from determinations at 6 or more pNPP concentrations).
Figure 4:
Inhibition of the
K-pNPPase activity of soluble protomeric
Na,K-ATPase by TNP-ADP. Data in the form of Dixon plots. A,
control enzyme; B, FITC-treated enzyme. In A, the
K
-phosphatase activity was assayed at the
TNP-ADPNa
concentrations shown and at 2 (solid
circles), 4 (open circles), and 6 mMpNPP (squares) and in B, at 3 (solid circles), 5 (open circles), and 9 mMpNPP (squares). The total sodium concentration
was kept constant. After solubilization, the FITC-treated enzyme
presented 88.6 ± 3.6% of the control
K
-phosphatase specific activity and 10.7 ± 1.6%
of the control Na,K-ATPase specific activity. The kinetic parameters of
the K
-phosphatase reaction of the
protomers
were (K
, mM, and V
,
µmol
min
mg
): 0.99
± 0.16 and 2.12 ± 0.06, for the control enzyme, and 3.84
± 0.12 and 1.88 ± 0.05, for the FITC-modified enzyme. The arrows in B indicate, for each pNPP
concentration, the maximal level of 1/v expected had only the
native component of the K
-phosphatase activity been
inhibited by TNP-ADP.
Figure 2:
Block of individual sodium pump protomers
by FITC. K-activated phosphatase activity (V
), Na,K-ATPase activity, and protein
concentration were measured in FITC-treated and control sodium pump, in
their original membrane-bound state (MB, stippled bars) and in
the
protomers arising after solubilizing with
C
E
(
, solid bars). The
specific activities of the FITC-treated enzyme are presented as percent
of the specific activities of the respective control enzyme. Open
bar, rescue of Na,K-ATPase specific activity to be expected after
solubilization, had FITC inactivated only one-half of a functional
(
)
membrane dimer. At 20 °C, the control
K
-phosphatase specific activities
(µmol
min
mg
) of
the membrane-bound and solubilized enzymes were 3.41 ± 0.08 and
1.28 ± 0.02, and the control Na,K-ATPase specific activities,
2.68 ± 0.11 and 1.54 ± 0.02, respectively. The initial
treatment was done for 3 h, at 2.4 mg of protein/ml, and with or
without 30 µM FITC, as detailed under ``Experimental
Procedures.''
Figure 3: Remaining Na,K-ATPase activity of soluble protomeric sodium pump, plotted against the activity of the respective membrane-bound enzyme, in independent FITC treatments like that of Fig. 2and to various inactivation levels. The solid line represents the least squares linear regression on the data. The lower dashed line shows the correlation anticipated for separate pNPP and FITC binding sites in each protomer; the upper dashed line sets out the expectation for a single, adaptable binding site per protomer if only one of the two protomers could bind FITC covalently in a functional membrane dimer. All vertical and horizontal bars represent ± S.E. As in Fig. 2, the errors have been compounded from the specific activity errors of the FITC-treated and control enzymes.
In
order to test for independent high affinity and low affinity ATP sites,
we wished to find out whether the K-phosphatase
activity of well-defined
protomers was inhibited by
nucleotides when the high-affinity ATP site had been blocked by the
modified fluorescein. The limited thermal stability of the solubilized
protomers, even at 20 °C(30) , thwarted attempts at
treating the enzyme with FITC at alkaline pH after detergent
solubilization; lower pH values would only prolong the incubation
period. The alternative was to solubilize the FITC-treated membrane
enzyme. It has been reported that C
E
solubilization of the FITC-modified sodium pump releases
particles (30) whose sedimentation coefficient (s
) is 6.9 S, i.e. as
expected for the
protomer(2, 26) . The
results presented in Table 1average to an identical
s
value and confirm that, in our hands also,
solubilization of the FITC-treated enzyme leads to
protomeric particles.
Before opting for the approach of solubilizing
after FITC treatment, however, certainty was needed that the high
affinity ATP site of every protomeric unit was occupied by
FITC when complete inactivation of the overall sodium pump reaction had
been achieved. The degree of confidence was not as high as desirable
for the present purpose (see ``Discussion''), and the
endurance of the K
-phosphatase activity in the
FITC-inactivated membrane enzyme seemed open, therefore, to two
alternative interpretations. One possible explanation was that, in each
protomeric unit, there was a site that bound the phosphatase
substrate that was different from the high affinity ATP site (which was
blocked by FITC). In this case, solubilization of the membrane-bound,
FITC-modified sodium pump with C
E
would
release
protomers whose Na,K-ATPase specific activity
(relative to the FITC-free control enzyme) was no more, and no less,
than the (relative) Na,K-ATPase specific activity of the parent
membrane-bound enzyme. The alternative possibility was that the high
affinity ATP site and the pNPP-binding site were
topographically one and the same, the different catalytic properties
arising from different enzyme conformations in the presence of
Na
or K
ions (31) . If the
membrane-bound sodium pump behaved as a dimer of interacting
protomers in these conditions, one could hypothesize that only one of
the protomers in the pair bound FITC covalently at its single site.
Although the FITC-blocked protomer should still be able to adopt
E
and E
conformations reversibly(20) ,
the unliganded protomer in the dimer might become locked in an
E
conformation, because of subunit interactions. In that
case, its K
-phosphatase activity would be spared but
ATP hydrolysis, which requires cyclical E
-E
transitions, would be prevented just as in the FITC-blocked
protomer. As solubilization with C
E
, in our
conditions, dissociates any (
)
dimers(2, 26) , the good protomer should now be
unencumbered by its FITC-blocked neighbor, and its Na,K-ATPase activity
would be restored. Accordingly, the specific Na,K-ATPase activity of
the soluble protomers arising from the FITC-modified enzyme should be
higher than that of the parent membrane-bound enzyme, by a magnitude
equal to half the loss observed in the latter.
Fig. 2shows
the result of the experiment to decide between the alternative
hypotheses. This was conducted with side-by-side FITC-free control
enzyme samples. Na,K-ATPase and K-phosphatase
activities, as well as protein concentration, were determined in both
inactivated and control samples, before and after solubilization, and
the specific activities (in
µmol
min
mg
)
calculated. The data represent 100 times the specific activity of the
FITC-modified enzyme, divided by the relevant (ATPase or phosphatase)
specific activity of the appropriate (membrane-bound or solubilized)
control enzyme. In agreement with previous observations, and in spite
of the very low level of Na,K-ATPase activity left over, the
K
-phosphatase activity remains at a high 80% in both
the membrane-bound and solubilized enzymes. The crucial feature,
however, is that the percent Na,K-ATPase activity left in the soluble
protomers does not differ from the percent Na,K-ATPase activity left in
the parent membrane-bound enzyme. In the case of the
``one-site-plus-dimer'' hypothesis, one should have expected
that the Na,K-ATPase increase to 51.6 ± 6.5% of the controls,
after solubilization (clear plus filled bars on right). The experiment was repeated at several levels of
inactivation of the Na,K-ATPase activity, and the result is shown in Fig. 3. It is apparent that, despite some dispersion in the
data, the results are firmly anchored on the 1:1 correlation line
expected for the ``two-sites-per-protomer'' hypothesis.
Table 2shows that the protomeric FITC-enzyme can efficiently
utilize the bulky 3-OMFP as a K-phosphatase substrate;
this makes it quite unlikely that the phosphatase-binding site be but a
small region of a single ATP-binding site. In order to test the
possibility that high ATP concentrations could overcome the FITC block
of the overall reaction, the Na,K-ATPase activities of FITC-modified
and control enzymes were measured in one experiment at 2 and 20 mM [
-
P]ATP. The specific activities of
the FITC-modified enzyme (relative to the control enzyme) were 0.9
± 0.1% at 2 mM ATP and 1.1 ± 0.1% at 20 mM ATP.
It being clear that FITC can block an ATP site in every
protomeric unit of the membrane-bound enzyme, we tested the effect of
nucleotides on the K-phosphatase activity of the
soluble protomers. Experiments using ATP (Tris salt) to 5 mM (not shown) produced clear effects with the control membrane-bound
and solubilized enzymes, but its effect on the FITC-modified enzyme
seemed to be obscured by a much reduced affinity (cf. (19) and (21) ). Lower pNPP or higher ATP
concentrations imposed unacceptably high spectrophotometric errors or
ionic strength compensations, respectively, and we resorted to using
TNP-ADP, which has proved useful in experiments with the membrane-bound
FITC-modified enzyme(19) . The result of one of three similar
experiments is presented in Fig. 4, as Dixon plots, for the
soluble protomers. Here, again, the affinity is very much reduced in
the FITC-treated protomers. K
works out as 65
± 8 µM, compared with the native protomers at 0.084
± 0.003 µM, but it is evident that TNP-ADP inhibits
the K
-phosphatase activity competitively well beyond
what could be expected if it merely suppressed the 10% of the sodium
pump that escaped FITC modification (arrows). Dixon plots of
the K
-phosphatase activity of the parent
membrane-bound FITC-modified enzyme (not shown) also reflected the
decreased TNP-ADP affinity, as has been observed earlier(19) ,
with K
values of 42 ± 9 µM and
0.52 ± 0.05 µM for the FITC-treated and control
enzymes, respectively. TNP-ATP was also effective with the
FITC-modified membrane enzyme, but the estimated inhibition constant
was 4-fold that for TNP-ADP, and it was thereby not used with the
soluble protomers. Picrate (2,4,6-trinitrophenolate) at 500 µM inhibited the K
-phosphatase activity of the
FITC-modified enzyme by 13% in conditions that 250 µM TNP-ADP caused a decrease of over 70%. It seems safe to conclude,
therefore, that the bulk of the TNP-ADP inhibition does not represent
nonspecific effects of the trinitrophenyl group.
Our test for a discrete low affinity nucleotide site in
Na,K-ATPase is based on searching for a known E effect of
nucleotides on
protomers with a blocked high affinity ATP
site. We chose FITC as the blocking agent and the
K
-phosphatase activity as the E
function.
The covalent FITC binding to Lys
suppresses
phosphorylation by ATP and the overall pump reaction, but does not
prevent backwards phosphorylation by inorganic phosphate or abolish the
K
-phosphatase activity of the enzyme. Obviously, the
possibility had to be excluded that any observable low affinity
nucleotide effects could arise from binding at a unique nucleotide site
that was left vacant in a neighboring and interacting subunit.
It
has been reported that, at 100% inactivation of the Na,K-ATPase
activity, the protein mass that can incorporate 1 mol of FITC can
otherwise bind 1 mol of ouabain (32) or form 1 mol of
acid-stable phosphoenzyme(33) . Our difficulty with this
approach was that, with the best preparations of native enzyme, it had
not been possible to achieve acid-stable phosphorylation or ouabain
binding levels of 1 mol/mol protomeric unit (6.9 nmol/mg of protein)
using the same standard phosphorylation or ouabain binding
assays, and that this did not seem just a matter of protein assay
method(34) . For instance, the standard ouabain/phosphorylation
ratio is subject to some uncertainty(35, 36) , and it
is now apparent that, depending on the experimental conditions,
measurements of acid-stable phosphorylation can grossly underestimate
the number of active sodium pump units(37, 38) . In
addition, it has been reported that FITC can also bind to Lys on the
chain, which is not necessarily within the high
affinity ATP-binding pocket(39) . Any one, or a combination, of
these complications could cause an overestimate of the number of
functional high affinity ATP sites that are occupied by FITC. This
possibility seemed the more plausible when considering the evidence
that the membrane-bound Na,K-ATPase behaved, at least in some
conditions, as a functional dimer of
protomers(7, 18, 40, 41, 42, 43) .
The concerns above prompted the experiments shown in Fig. 2and Fig. 3, which exclude the possibility that only
one-half of a putative membrane dimer could bind FITC. If all active
protomers in the membrane-bound enzyme are potential targets
for the binding, the corollary would be that the
K
-phosphatase substrate site should be different from
the blocked high affinity ATP site. It would follow also that the
competitive TNP-ADP inhibition of the K
-phosphatase
activity of FITC-modified soluble protomers (Fig. 4) should
result from TNP-ADP binding at a location different from the blocked
site, probably at the pNPP-binding site. We have confirmed
that, in the membrane-bound enzyme, FITC treatment decreases the
affinities of pNPP, ATP, and TNP-ADP for their E
effects (19, 21) and find that this effect
persists after solubilization to
protomers. In the control
enzyme, this decreases K
for TNP-ADP from 0.52
µM (cf. (17) ) to 0.084 µM,
which agrees with the 8-fold decrease of K
observed for the low affinity ATP activation of the Na,K-ATPase
activity upon solubilization (2) . This downward shift did not
occur with the FITC-modified enzyme, as K
changed
from 42 µM in the membrane-bound enzyme (compare with 35
µM in (19) ) to 65 µM after
solubilization. The possibility should be considered, therefore, that
all that FITC does is greatly lowering the affinity of two states of a
single ATP site. If that were so, the FITC block of the Na,K-ATPase
activity should be relieved by increasing the ATP concentration. This
did not occur at 8 mM ATP when using the coupled enzyme
spectrophotometric assay(19) , and we now find that it does not
happen at 20 mM [
-
P]ATP either.
Had FITC lowered both high and low affinities by, say, a thousandfold
(so that K
200 mM), we would
have easily noticed an increase from 1% (at 2 mM) to 9% (at 20
mM) of the control Na,K-ATPase activity. By far, the most
likely explanation is that ATP is utterly prevented from accessing the
catalytic pocket by the bound fluorescein which also induces a
distortion in the low-affinity
nucleotide/K
-phosphatase site. The block of the
catalytic ATP site probably results from the restricted rotational
motion of the bound fluorescein, as observed even through
E
-E
conformational changes(44) .
It
is conceivable that the catalytic ATP site and the phosphatase site
could share binding groups, perhaps their phosphate subsites. At any
rate, such overlap must be such that access be still allowed to the
fluorescein moiety of 3-OMFP when another fluorescein blocks the
catalytic ATP site of the FITC-enzyme (Table 2). On the other
hand, it seems probable that the second nucleotide site be the same as
the phosphatase site, a question that can be explored in more elaborate
experiments. Fully competitive inhibition (same site) of the
K-phosphatase by nucleotides should lead to linear
Dixon plots whereas partially competitive (allosteric) inhibition
should show as hyperbolae(45) . The plots in Fig. 4look
convincingly linear, but higher inhibitor and lower substrate
concentrations might lead to a curvature(46) . Downward-bent
Dixon plots do appear when using ATP as the inhibitor with the
membrane-bound sodium pump(21) , and we have confirmed this,
also with the
protomers. Caution is needed in their
interpretation, though, as in a partially modified enzyme the
difference in kinetic parameters between FITC-inactivated and
outlasting enzyme populations could lead to an artifactual downward
curvature. For instance, had the FITC-modified enzyme been impervious
to TNP-ADP inhibition, the plots in Fig. 4would have bent down
and become parallel to the abscissa, at maximal ordinate levels given
by the arrows in panel B (and at much lower
values of the abscissa, compare with panel A).
The
test of Fig. 2and Fig. 3does not contradict the
possibility that the membrane-bound sodium pump behave, at least in
some conditions, as a dimer of and protomers. What it proves
is the absence of half-of-the-sites reactivity toward FITC. However,
some of the evidence for a dimeric Na,K-ATPase may need reinterpreting
in consideration of a second nucleotide site per protomeric unit, and
the added element of complexity should be taken into account. The
functional relationships between the two nucleotide sites, and their
behavior during the reaction cycle, remain to be explored.