From the Transport ATPase Laboratory, Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, University of Leicester, Leicester LE1 9HN, United Kingdom
Received for publication, October 30, 2002, and in revised form, February 13, 2003
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ABSTRACT |
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The Na+-dependent
or E1 stages of the Na,K-ATPase reaction require a few micromolar ATP,
but submillimolar concentrations are needed to accelerate the
K+-dependent or E2 half of the cycle. Here we
use Co(NH3)4ATP as a tool to study ATP sites in
Na,K-ATPase. The analogue inactivates the K+ phosphatase
activity (an E2 partial reaction) and the Na,K-ATPase activity in
parallel, whereas ATP-[3H]ADP exchange (an E1
reaction) is affected less or not at all. Although the inactivation
occurs as a consequence of low affinity Co(NH3)4ATP binding (KD The sodium pump or
Na,K-ATPase1 is the enzyme
that mediates active transport of Na+ and K+
across the plasma membrane of animal cells (1-3). It consists of an
Both transport and ATP hydrolysis by Na,K-ATPase have complex responses
to ATP (12, 13). This behavior is also observed with the Several laboratories have suggested the co-existence of separate high
affinity and low affinity ATP sites in P-type ATPases (for review,
see Ref. 21). One of the paradigms is that of FITC, which binds
covalently to The experiments in the present paper make use of
Co(NH3)4ATP, a substitution-inert ATP analogue
(31-34) with actions complementary to those of FITC. Following
equilibrium binding at a low affinity site,
Co(NH3)4ATP becomes trapped, albeit
non-covalently, and inactivates Na,K-ATPase by blocking E2 reactions
but not E1 steps (31, 32). Interestingly, after incorporating a maximal
quantity of Co(NH3)4[ Enzyme Purification--
Na,K-ATPase was purified in
membrane-bound form from pig kidney outer medulla, by using the
zonal-rotor procedure of Jørgensen (35), and stored at 0 °C in 25 mM imidazole, 1 mM EDTA (pH 7.4). The specific
activity ranged from 20 to 35 µmol min Co(NH3)4ATP
Synthesis--
This was carried out according to Cornelius et
al. (36). The purity of the product was assessed by high
performance liquid chromatography on a Partisil 10 SAX column (see
under "ATP-ADP Exchange"), where
Co(NH3)4ATP eluted at 4:24 min (Fig.
1A). The elution time is
similar to that of AMP (Fig. 1B), as expected for the
bidentate complex (37). The concentration of the product was assessed
from the UV and visible spectrum and the molar extinction coefficients
at 257, 366, or 518 nm (36).
Co(NH3)4[3H]ATP was synthesized
from [2,8-3H]ATP in the same manner, to specific
activities between 500 and 3000 MBq mmol Inactivation of Na,K-ATPase with
Co(NH3)4ATP--
Aliquots of
purified Na,K-ATPase (at about 1 mg of protein ml Inactivation with FITC--
This was done essentially as
described (29), at protein concentrations between 1 and 2.5 mg/ml and
in a medium containing 150 mM NaCl, 50 mM
Tris/Cl Equilibrium Binding of
Co(NH3)4[3H]ATP
or [ Na,K-ATPase Solubilization to Analytical Ultracentrifugation of the Solubilized
Enzyme--
Sedimentation velocity runs were carried out at 40,000 rpm
and 20 °C on a Beckman Optima XL-A analytical ultracentrifuge (6) (National Centre for Macromolecular Hydrodynamics, Leicester and Nottingham Universities, UK). The enzyme was solubilized with C12E8 as described above, and the high speed
supernatants of control and
Co(NH3)4ATP-inactivated enzymes were analyzed
in the same run. The double-sectored cells were scanned radially at 280 nm at 20- or 30-min intervals; all samples showed a single transition zone. The sedimentation coefficient
s20,b (at 20 °C in buffer) and
its intrinsic error were calculated from linear regression on the
natural logarithm of the radial distance of the midpoint of the
depletion region versus time and corrected to
s20,w (39). The protein molecular mass
of the solubilized enzyme was calculated according to Tanford et
al. (40), using a Stokes radius of 72 Å and a partial molar
volume = 0.646 cm3 g Na,K-ATPase Determinations--
ATP hydrolysis was measured from
the release of 32Pi from
[ K+ Phosphatase Activity--
This was evaluated in
either of two ways. To measure the release of p-nitrophenol
from pNPP (29) at 20 or 37 °C, the medium routinely
contained (mM) the following: pNPP 10, MgCl2 6, KCl 150, imidazole 20 (pH 7.2), plus enzyme at
20-30 µg ml ATP-ADP Exchange--
The rate of reversible initial
phosphorylation of Na,K-ATPase was measured at 20 °C from the
backward half-reaction, as incorporation of the
[2,8-3H]ADP label into ATP, and was corrected for
[ Protein Determinations--
Protein was determined in 0.5-ml
samples, at least in triplicate, using a modified bicinchoninic acid
method (6, 42) with bovine serum albumin as standard.
Source of Materials--
ATPNa2 and ADP (free acid)
were from Roche Molecular Biochemicals; [2,8-3H]ATP,
[2,8-3H]ADP, [ Data Handling--
In Na,K-ATPase and K+ phosphatase
assays, the reaction time courses were fitted by least squares linear
regression to obtain the enzyme activities and their standard errors.
The calculation of the rate of ATP-ADP exchange took the fractional ATP
hydrolysis into account; the rate constant for the reverse
half-reaction was obtained from the time course data, fitted as a
linear transform for exponential approach to isotopic equilibrium (41).
Where relative (or percent) specific activities are shown, the errors have been compounded to incorporate the error on the specific activity
of the reference sample. Other linear and non-linear curve fitting were
done using SigmaPlot 4 (Corte Madera, CA).
Co(NH3)4ATP-binding
Sites--
Two characteristic features of the
Co(NH3)4ATP inactivation of Na,K-ATPase are the
selective suppression of E2 reactions and the distinctive low affinity
of the Co(NH3)4ATP binding step preceding it
(31, 32). The experiment in Fig. 2
confirms the first of these attributes. Here we have measured the
overall reaction (Na,K-ATPase activity) and two partial reactions,
ATP-[3H]ADP exchange (an E1 reaction) and the
K+ phosphatase activity (an E2 reaction).
ATP-[3H]ADP exchange results from a reversal of the
initial enzyme phosphorylation, as the 3H label becomes
distributed into the ATP compartment. It is apparent that when the
membrane-bound sodium pump is exposed to
Co(NH3)4ATP at 37 °C, and then washed by
ultracentrifugation, it loses its K+ phosphatase and
Na,K-ATPase activities at the same pace. In our hands, the ATP-ADP
exchange activity of the Co(NH3)4ATP-treated enzyme returns somewhat variable results, but its survival value is
always substantially higher than those of the K+
phosphatase or Na,K-ATPase activities. In the experiment shown in Fig.
2, there was no loss of exchange by the time the other two activities
had fallen to a few percent of their controls. Fig.
3A shows how the pseudo-first
order rate constant for inactivation of the Na,K-ATPase activity
increases with the Co(NH3)4ATP concentration. The hyperbolic dependence confirms that this is a two-step process (31,
38), i.e. that saturable low affinity
Co(NH3)4ATP binding at a single enzyme site
must occur before Co(NH3)4ATP occlusion and
enzyme inactivation. A dissociation constant of 0.62 mM
could be fitted to the data, similar to the value reported earlier
(31). This is in the concentration range for low affinity ATP effects on the sodium pump, as found when measuring Na,K-ATPase activity (13)
or E2 reactions like K+:K+ exchange (18, 44)
and K+ (Rb+) occlusion (15).
It has also been reported that Co(NH3)4ATP can
act as a competitor in experiments to measure high affinity equilibrium
ATP binding (31, 33) but without permanent deleterious effects on the
enzyme. We have now made direct measurements of high affinity equilibrium Co(NH3)4ATP binding; the
experiments shown in Fig. 3B demonstrate that, with
KD = 0.10 µM,
Co(NH3)4ATP is as good a ligand as ATP in these
conditions (cf. Ref. 45) if not better. Incidentally, given
the concentration dependence and temperature in Fig. 3A, and
as either Na+ or K+ inhibit the enzyme
modification by Co(NH3)4ATP (31), in the equilibrium binding experiments of Fig. 3B (at 20 °C)
there must have been little or no enzyme inactivation. To summarize,
one may conclude that, with non-cycling Na,K-ATPase, it is
possible to observe high affinity Co(NH3)4ATP
binding directly and low affinity Co(NH3)4ATP
binding at least indirectly.
Complementary Co(NH3)4ATP
and FITC Modifications--
The primary
Co(NH3)4ATP interaction with the K+
phosphatase reaction is such that the analogue behaves as a competitive
inhibitor with respect to pNPP, with Ki = 0.22 mM (33). We have now looked at the consequences of
Co(NH3)4ATP inactivation. The experiment shown
in Fig. 4A compares the
pNPP concentration dependence before and after the enzyme
was inactivated with, and washed free from,
Co(NH3)4ATP. Whereas
K0.5 shows no increase, the maximal K+ phosphatase rate has been reduced considerably, to
12 ± 2%. This is not too different from the residual level of
Na,K-ATPase activity and suffices to explain the parallel inactivation
time courses seen in Fig. 2. The obvious conclusion is that, rather
than reducing the pNPP affinity, the
Co(NH3)4ATP modification fully prevents the
K+ phosphatase domain from binding or hydrolyzing its
substrate. As the irreversible effect is preceded by low affinity
Co(NH3)4ATP binding to the enzyme, and as the
ATP-ADP exchange activity is unaffected (Fig. 2), a plausible
conclusion is that the low affinity Co(NH3)4ATP-binding site and the K+
phosphatase site are different from the site that supports enzyme phosphorylation by ATP, a notion that is reinforced by the results shown in Fig. 5. Fig. 4B shows
a contrasting experiment, carried out with FITC-modified Na,K-ATPase.
The FITC modification increased K0.5 for
pNPP about 2-fold but the maximal K+ phosphatase
rate was more or less unaffected, as reported previously (26, 29). The
FITC modification does not hinder 32Pi
phosphorylation either, but it prevents high affinity ATP binding and
phosphorylation by micromolar [
The complementarity of Co(NH3)4ATP and FITC
modifications had suggested that the enzyme could accommodate both
ligands at the same time, in different pockets (47). We treated the
enzyme with FITC until its Na,K-ATPase activity had been reduced to 1% that of a parallel control enzyme; its K+ phosphatase
activity, measured with pNPP, remained at 75%. When the
FITC-modified and control enzymes were incubated side by side with 1 mM Co(NH3)4ATP, the rate constants
for inactivation of their K+ phosphatase activities were
0.75 ± 0.02 and 0.86 ± 0.01 h The Number of ATP Sites Per Protomeric Unit--
The above
experiments support the view that two ATP-binding sites co-exist in
Na,K-ATPase (6, 21, 27, 29-31). The controversial question has been
whether this means that there is one high affinity and one low affinity
ATP site per
If the protomeric unit had one high affinity and one low affinity site
and Co(NH3)4ATP only blocked the latter, the
percent Na,K-ATPase activity left in the inactivated soluble protomers should be identical to that of the inactivated membrane-bound enzyme,
and the same should apply to the K+ phosphatase activity.
On the other hand, if single-site protomers were organized as an
( Direct Co(NH3)4ATP
Inactivation of Solubilized Co(NH3)4ATP and FITC as ATP
Site Probes--
As a first step, we confirmed that it was possible to
measure ATP-[3H]ADP exchange following low affinity
Co(NH3)4ATP inactivation of E2 functions and
turnover (Fig. 2) (32). Although the dephosphorylating activity had
been blocked, the minimal enzyme unit retained functional phosphorylation and high affinity nucleotide sites, as well as the
catalytic machinery and conformational freedom needed for reversible
kinase activity.
The results in Fig. 3 show that high and low affinity
Co(NH3)4ATP binding are properties
inherent to the native non-cycling Na,K-ATPase. Complexation of ATP by
the Co(III)(NH3)4 group does not lead to
deleterious effects on high affinity binding to the enzyme (Fig.
3B), and it might even lower KD values. It is also clear, and despite suggestions to the contrary (51), that
low affinity nucleotide binding (Fig. 3A) is not the result of previous FITC modification nor the kinetic outcome of a branched reaction cycle. All the same, Fig. 5 shows that
Co(NH3)4ATP can also bind to the FITC-modified
enzyme, with low affinity and without hindrance. The ensuing
Co(NH3)4ATP trapping inactivates the
K+ phosphatase activity that had survived the FITC
treatment and not only does the low Co(NH3)4ATP
binding affinity remain unaltered, but there is also little change in
the maximal inactivation rate constant. Evidently, the occluded
Co(NH3)4ATP state is being reached with a
probability similar to that in the native enzyme, and this suggests
that the conformational flexibility around this extant low affinity
site is preserved after FITC modification of the enzyme.
Two Sites Per Protomer or a Dimeric Membrane Enzyme?--
If there
were a single ATP site per
The demonstration above rests on the assumption that the enzymic
reactions catalyzed by the solubilized enzyme genuinely represent the
activity of the
Another explanation has been suggested (48) for results like those in
Figs. 6 and 7, that a very small dimer population that would escape
detection during analytical ultracentrifugation could exist in rapid
equilibrium with the protomer. That dimer, and not the protomer, would
be the only soluble particle with enzymic activity or would at least be
the source of non-Michaelian ATP effects. Apart from being kinetically
unlikely (6), this possibility is excluded by the very same results in
Fig. 7 (also see Fig. 3 of Ref. 29). The reason, again, is that if only
one Direct Co(NH3)4ATP
Inactivation of the Soluble Evidence Against Two ATP Sites--
In a study with enzyme
purified from duck nasal gland (51), the Na,K-ATPase and K+
phosphatase activities appeared to be equally inhibited by TNP-ADP, or
inactivated by increasing ErITC concentrations. However, the ErITC data
should be interpreted with caution as the inactivation was evaluated
only after stoichiometric completion had been reached, and this may
have concealed different ErITC concentration dependences of the rate
constants (38). At any rate, in the case of SERCA (55) ErITC
inactivates the low affinity ATP effect on the Ca-ATPase reaction far
more effectively than the high affinity effect. As the behavior of the
K+ phosphatase activity tends to line up with low affinity
ATP interactions and the Na,K-ATPase activity was only measured at high
ATP concentrations (51), the finding with the sodium pump might not be
addressing the relevant issue. On the other hand, with gastric
H,K-ATPase (56) there is an apparent and compound dilemma that TNP-ATP binds with a single affinity but a stoichiometry of 2 mol per phosphorylation site and that ATP competes the TNP-ATP binding with two
ATP dissociation constants differing by a factor of >300. This
illustrates the risk that even real affinities can be misleading if
used selectively and, in isolation, as ultimate evidence (a caveat that
cuts both ways, as discussed by Faller (56)).
A recent study (57) has reported results of site-directed mutagenesis
of residues lining a predicted nucleotide binding pocket in rat
The evidence against the existence of a regulatory ATP site is broadly
based on experiments that show that although binding at an apparently
single site, some ligands can inhibit more than one Na,K-ATPase
function or interfere with both high and low affinity ATP effects (51,
57, 58). Although these findings are interesting in their own right,
the possibility should be considered that some of these agents or
procedures act through global effects, the case of ouabain is a prime
example, or that the experimental conditions are unfavorable for the
question at hand (27, 58). In fact, the converse remains the crucial
quandary, i.e. that certain ATP analogues can selectively
inhibit or inactivate some partial reactions and not others or do so
with different affinities. For instance,
Cr(H2O)4-adenosine
5'-[ ATP Sites and the Tridimensional SERCA Structure--
On balance,
the results of this work support the notion of independent high and low
affinity ATP-binding sites in the protomeric unit. When considering
whether FITC and Co(NH3)4ATP might bind within
one broad nucleotide site or at two separate pockets, the crucial point
is that if the former is true, docking of these ligands at their
locations should be non-overlapping and apparently non-interacting.
Recent preliminary experiments measuring fluorescence energy transfer
return a Förster distance of 29 Å between FITC and the cobalt of
Co(NH3)4ATP bound at equilibrium to Na,K-ATPase (65, 66). In the high resolution open SERCA structure (24), this is
roughly the distance between Lys515, the anchoring point of
FITC in the N domain, and Asp351, the phosphorylation site
on the P domain, and by far exceeds the span of an ATP molecule. A
single nucleotide-binding site has been located in the N domain (24)
from the increased electron density obtained after diffusion of TNP-AMP
into the crystals. TNP-AMP is the nucleotide with the highest affinity
toward SERCA, and TNP-[3H]AMP binds at equilibrium with a
stoichiometry of 1 mol per phosphorylation site (67). However, although
2 mol of TNP-[3H]ATP can be bound per mol of SERCA in
similar conditions, and two competing ATP effects may be observed (67,
68), there have been no reports of a second ATP site in SERCA crystals.
As mentioned above, twice stoichiometric TNP-ATP binding and dual ATP
competition can also be seen with gastric H,K-ATPase (56).
Recent experiments (69, 70) have shown that
TNP-8N3[ 0.4-0.6 mM), we can also measure high affinity
equilibrium binding of
Co(NH3)4[3H]ATP
(KD = 0.1 µM) to the native enzyme.
Crucially, we find that covalent enzyme modification with fluorescein
isothiocyanate (which blocks E1 reactions) causes little or no effect
on the affinity of the binding step preceding
Co(NH3)4ATP inactivation and only a
20% decrease in maximal inactivation rate. This suggests that
fluorescein isothiocyanate and Co(NH3)4ATP bind
within different enzyme pockets. The
Co(NH3)4ATP enzyme was solubilized with
C12E8 to a homogeneous population of
protomers, as verified by analytical ultracentrifugation; the
solubilization did not increase the Na,K-ATPase activity of the
Co(NH3)4ATP enzyme with respect to parallel
controls. This was contrary to the expectation for a hypothetical
(
)2 membrane dimer with a single ATP site per
protomer, with or without fast dimer/protomer equilibrium in detergent
solution. Besides, the solubilized
protomer could be directly
inactivated by Co(NH3)4ATP, to less than 10%
of the control Na,K-ATPase activity. This suggests that the
inactivation must follow Co(NH3)4ATP binding at
a low affinity site in every protomeric unit, thus still allowing ATP and ADP access to phosphorylation and high affinity ATP sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or catalytic subunit (Mr 112,000) and a
subunit, a glycoprotein (protein Mr 35,000)
found in a 1:1 ratio (2). The
heterodimer, or
protomer,
appears as the minimal catalytically active unit after solubilization
with C12E8 (4-6) and probably also represents the minimal enzymic unit in the membrane (7, 8). There is general
agreement that the reaction cycle of Na,K-ATPase and other P-type
ATPases proceeds through the formation and breakdown of phosphoenzyme
intermediates (9, 10), as the enzyme cycles between an E1 or
Na+ form, and an E2 or K+ form (11).
protomer (6) and seems to result from two types of ATP interaction with
the enzyme. On the one hand, the E1
(Na+-dependent) reactions are saturated at a
few micromolar ATP and are associated with phosphorylation of the
-chain at Asp369 (2, 14). In the case of Na,K-ATPase,
the next documented ATP requirement only arises after hydrolysis of the
phosphoenzyme; it consists of an acceleration of K+ release
from the "occluded K+ form" to the intracellular
medium, a rate-limiting step (9, 15, 16), and results in a large
stimulation of pump activity. Here ATP acts with a low affinity, with
K0.5 in the region of 0.2-0.5 mM
(15), and can be replaced by ADP (17) and non-phosphorylating analogues
of ATP (18). The requirements and affinities of E1 and E2 reactions
largely explain the apparent negative co-operativity in the ATP
activation of Na,K-ATPase activity and Na+-K+
exchange. Nucleotides also show low affinity inhibitory effects toward
some E2 reactions of Na,K-ATPase. Among these are the hydrolysis of the
phosphoenzyme formed from 32Pi and the
K+ phosphatase activity, i.e. the ability to
hydrolyze synthetic substrates, like p-nitrophenyl phosphate
and 3-O-methylfluorescein phosphate, in the presence of
K+ (19, 20).
Lys501 in Na,K-ATPase (22) and
Lys515 in SERCA (23), in what now clearly appears as a high
affinity adenine-binding site; in the 2.6-Å SERCA structure (24)
Lys515 is located deep inside a nucleotide binding pocket
in the N domain. FITC blocks E1 functions like high affinity ATP
binding and [
-32P]ATP phosphorylation, as well as the
overall (Na,K-ATPase) activity (25-27). On the other hand, E2
functions like 32Pi phosphorylation and
dephosphorylation, and the K+ phosphatase activity, are
little affected by the FITC modification; the latter is true whether
the substrate is acetyl phosphate, pNPP, or 3-OMPF (25-29,
73). In the FITC-enzyme, both the K+ phosphatase activity
and dephosphorylation of the phosphoenzyme formed with
32Pi are inhibited by ATP and TNP-ADP, with low
affinities (28, 29). We have reported that FITC can access all
-chains of membrane-bound Na,K-ATPase, that pNPP and
3-OMFP can be split by the solubilized, FITC-modified
protomer,
and that TNP-ADP can inhibit and TNP-8N3-ADP photoinactivate the K+ phosphatase activity of the FITC
enzyme (29, 30). This suggested that TNP-ADP should bind at a low
affinity nucleotide site on the FITC-modified
-chain.
-32P]ATP
in the membrane enzyme, it was still possible to bind an equivalent
amount of Cr[
-32P]ATP but not
Co(NH3)4[32P]PO4
(34). Our results show that Co(NH3)4ATP can
access all
protomeric units, either in detergent solution or in
the membrane, and that the survival of E1 functions can be best
explained on the basis of separate high affinity and low affinity ATP
sites on the
-chain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 mg
protein
1.
1. The purified
products were stored in aliquots at
80 °C.
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Fig. 1.
HPLC of adenine nucleotides. The samples
were run on a Partisil 10 SAX column (0.46 × 25 cm) with A = 50 mM ammonium formate (pH 4.6) and B = 1.4 M ammonium phosphate (pH 3.7). The straight line
segments show the gradient in % B, recorded simultaneously
with offset compensation. A,
Co(NH3)4ATP. One ml was injected; the 260 nm
trace was obtained with a 20 µM sample and the 518 nm
trace with a 1 mM sample, and both runs were superimposed
on the same chart. B, AMP, ADP, and ATP. Twenty nmol of each
(1 ml) in 5 mM Tris/Cl (pH 7.5), monitored at 260 nm.
Horizontal bars show collection windows for the fractions
containing AMP + Pi (M), ADP (D), and
ATP (T).
1) were
incubated for up to 3 h at 37 °C in 20 mM imidazole
(pH 6.8), with up to 3 mM
Co(NH3)4ATP or without (controls). In most experiments, the enzyme samples were cooled down and centrifuged for 15 min at 425,000 × g and 5 °C in a Beckman TL100
benchtop ultracentrifuge. The membranes were resuspended in chilled 20 mM imidazole (pH 6.8) with the help of a Dounce
homogenizer, washed once by ultracentrifugation, and resuspended in the
same medium. Protein and enzymic activities were then determined in
inactivated and control suspensions. In the experiment shown in Fig. 5,
the incubation at increasing Co(NH3)4ATP
concentrations was made in 30 µl, and 4-µl aliquots were diluted
into 0.8 ml of K+ phosphatase medium (containing 3-OMFP) in
a spectrophotometer cuvette, and the K+ phosphatase
reaction was followed as described below. To analyze the
Co(NH3)4ATP concentration effect, the
inactivation rate constant kinact was first
calculated as the negative slope of plots of the natural logarithm of
the fractional activity left against time, and the results were
replotted against the Co(NH3)4ATP concentration (38).
(pH 9.2), 5 mM EDTA, and 0.5%
dimethyl sulfoxide. Parallel enzyme samples were incubated with or
without 20 µM FITC for up to 4 h at 20 °C, spun
down, washed, and resuspended with 20 mM
Tris/Cl
(pH 7.5), 2 mM EDTA, 2 mM
dithiothreitol, or with 40 mM imidazole (pH 6.8), 2 mM EDTA.
-33P]ATP--
Equal volumes of a Na,K-ATPase
suspension and radioligand solution were mixed and incubated with
stirring for 20 min at 20 °C so that the final concentrations
were as follows (mM): NaCl 150, TES 10 (pH 7.2),
dithiothreitol 1, EDTA 1. At the end of this period, a volume was
extracted by means of a syringe (27) fitted with a µStar®
cellulose-nitrate filter (Costar, 0.2-µm pore diameter). One ml of
the filtrate ("free nucleotide") was counted in a Beckman
LS-6000-TA scintillation spectrometer side by side with 1 ml of the
suspension ("total nucleotide"), with quench, chemiluminescence,
and decay corrections and against calibrated Co(NH3)4[3H]ATP or
[
-33P]ATP standards. Bound nucleotide was calculated
from total and free nucleotide measurements and referred to the protein
mass of the sample.
Protomers--
The purified
enzyme was solubilized as already described (4-6). Briefly, samples of
control and Co(NH3)4ATP-treated Na,K-ATPase were washed by ultracentrifugation and resuspended with a medium containing 300 mM NaCl and 40 mM TES (adjusted
to pH 7.4-7.5 with NaOH). The protein concentration was determined and
then adjusted to 680 µg ml
1; an equal volume of
C12E8 solution was added to achieve a final detergent concentration of 1 mg ml
1. After 30 min at room
temperature, the samples were ultracentrifuged at 435,000 × g for 15 min and the pellets discarded. Enzymic assays were
performed as described below, except that C12E8
was added at the critical micelle concentration (50 µg
ml
1) to all reaction media.
1 (4, 5).
-32P]ATP, as described previously (6). All enzymic
assays were routinely conducted at 20 °C and as time courses with at
least 6 time points over 10 min. Na,K-ATPase reactions were carried out
in 50-µl samples of a medium containing (mM) the
following: [
-32P]ATP 1, MgCl2 1.5, NaCl
130, KCl 20, imidazole 20 (pH 7.2), plus membrane-bound or soluble
enzyme at 20-30 µg ml
1. The reactions were started by
mixing components pre-equilibrated at 20 °C and stopped by immersing
samples in a dry ice/ethanol bath. After defrosting at 0 °C,
32Pi was extracted as phosphomolybdate into
isobutyl alcohol/ethyl acetate (1:1), side by side with
acid-hydrolyzed standards, and aliquots were counted in a scintillation spectrometer.
1 (the pNPP concentration was
varied in the experiments shown in Fig. 4). Reaction samples (50 µl)
were stopped by dilution into 300 µl of 0.1 M NaOH, and
the absorbances were read at 410 nm in a Beckman DU65
spectrophotometer against a p-nitrophenol calibration curve.
In the experiment shown in Fig. 5, instead, the medium above contained
0.5 mM 3-OMFP instead of pNPP, and the release of 3-OMF was followed for up to 300 s at 475 nm and 37 °C. This was done in thermostatted, stirred 1-ml cuvettes in a U-3310 Hitachi spectrophotometer fitted with a 6-cell changer (cycling time, 30 s; sampling time, 3 s). All measurements were corrected for spontaneous 3-OMFP hydrolysis. As with pNPP, all phosphatase
activities were obtained from linear regressions on at least 6 time
points of product release against time.
-32P]ATP hydrolysis (41). The reactions were
generally carried out in a medium containing (mM) the
following: [2,8-3H]ADP 1.25, [
-32P]ATP
1, MgCl2 1.6 (400 µM calculated
Mg2+), NaCl 10, imidazole 20 (pH 7.2). Samples (10-20
µl) were stopped by mixing with 5 µl of 120 mM EDTA at
room temperature and immersing in a dry ice/ethanol bath. Defrosted
samples and "initials" were mixed with chilled
ATP/ADP/AMP/Pi carriers, and the nucleotides and
Pi were separated on a Partisil 10 SAX column (25 × 0.46 cm), using an Amersham Biosciences HPLC system (Fig.
1B). The separation was optimized for base-line resolution
and speed, with an elution rate at 1.5 ml min
1, and the
following gradient (min:s/% B): 0:00/0%, 0:01/15%, 5:40/50%, 5:45/100%, 16:00/100%, 16:01/0%, and 22:01/0%; where A is 50 mM ammonium formate (pH 4.6) and B is 1.4 M
ammonium phosphate (pH 3.7). The elution times (and collection times in
parentheses) in min:s are as follows: AMP/Pi 4:27
(3:55-5:55), ADP 6:26 (5:55-7:55), and ATP 9:25 (8:45-10:45). The
eluted fractions (3 ml), plus 12 ml of scintillation fluid
(Quicksafe-A, Zinsser Analytic) and 3 ml ethanol (41), were counted for
3H and 32P with quench, decay, and
chemiluminescence corrections, together with standards and
initial samples.
-32P]ATP, and
[
-33P]ATP were purchased from PerkinElmer Life
Sciences, and FITC was from Molecular Probes or Sigma.
[Co(III)(NH3)4CO3]NO3
was synthesized according to Schlessinger (43). Bicinchoninic acid was
from Pierce. HPLC columns were purchased from BASTechnicol (Stockport,
Cheshire, UK). pNPP (Tris salt), 3-OMFP, and 3-OMF were from
Sigma. All other reagents were of the maximal purity available.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Selective Co(NH3)4ATP
inactivation of E2 function of membrane-bound Na,K-ATPase. Eight
enzyme samples were incubated in 1 mM imidazole (pH 6.8)
for a total of 3 h at 37 °C, and
Co(NH3)4ATP was added to a final concentration
of 1 mM, timed to achieve the inactivation periods shown.
The samples were cooled down, centrifuged (15 min at 435,000 × g), washed once, and resuspended with 1 mM
imidazole (pH 6.8), and their protein concentration and enzymic
activities were determined. The plots show the Na,K-ATPase (open
circles), K+ phosphatase (solid circles),
and ATP-ADP exchange activities (squares), all assayed as
time courses at 20 °C, as described under "Experimental
Procedures"; vertical bars show standard errors. The
control values were (milliunits mg 1 ± S.E.) 1590 ± 40 (Na,K-ATPase), 536 ± 18 (K+ phosphatase), and
4.42 ± 0.06 (ATP-ADP exchange).
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Fig. 3.
High and low affinity
Co(NH3)4ATP binding. A,
Co(NH3)4ATP concentration dependence of the
rate of inactivation of membrane-bound Na,K-ATPase. The enzyme was
preincubated at 37 °C in 30 mM imidazole (pH 7.2), 1 mM EDTA, with or without
Co(NH3)4ATP at 8 concentrations between 1 µM and 3 mM, and 5 timed samples were drawn
within 30 (below 30 µM) or 20 min (between 0.1 and 3 mM). In all 8 × 5 resulting reactions, the
Na,K-ATPase activity was determined as linear time courses, and
inactivation rate constants kinact were
calculated from semilog plots of activity remaining versus
time of incubation with Co(NH3)4ATP. The
Co(NH3)4ATP concentration dependence of
kinact was fitted to the hyperbola shown, with
KD = 0.62 ± 0.11 mM and
kinact(max) = 2.40 ± 0.18 h 1
(mean ± S.E.). B, concentration dependence of high
affinity Co(NH3)4[3H]ATP and
[
-33P]ATP equilibrium binding to native Na,K-ATPase.
In separate experiments, the enzyme (15 µg ml
1) was
equilibrated with various concentrations of the radioactive nucleotide
at 20 °C, and in a medium containing (mM) NaCl 150, TES
(pH 7.2) 10, dithiothreitol 1, EDTA 1. The total and free labeled
nucleotide concentrations were determined as described under
"Experimental Procedures"; the bound nucleotide on
left-hand and right-hand ordinates are referred
to the protein concentration. In experiment 1 (solid circles,
long-dashed line, left-hand axis), the hyperbola
B(ound) = Bmax/(1 + KD/[free]) was fitted to the data, with
Bmax = 1.80 ± 0.22 nmol mg
1
and KD = 0.10 ± 0.02 µM; in
experiment 2 (open circles, solid line, left-hand axis), the
continuous line was fitted with Bmax = 1.75 ± 0.15 nmol mg
1 and KD = 0.11 ± 0.02 µM. For comparison, the lower curve (squares,
short-dashed line, right-hand axis) shows equilibrium
[
-33P]ATP binding to native enzyme, fitted with
Bmax = 1.47 ± 0.10 nmol mg
1
and KD = 0.24 ± 0.03 µM
(mean ± S.E.).
-32P]ATP (27, 28). The
covalently bound FITC would then be blocking the high affinity ATP
site, at least its adenine-binding region (46). Large ATP
concentrations during assays with the FITC-modified enzyme fail to
increase the low remaining level of Na,K-ATPase activity (28, 29).
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Fig. 4.
Effects of
Co(NH3)4ATP and FITC modifications on the
K+-phosphatase reaction. A, effect of
Co(NH3)4ATP inactivation of native sodium pump
on the substrate concentration dependence of pNPP
hydrolysis. Enzyme samples were incubated with or without 1 mM Co(NH3)4ATP for 3 h at
37 °C, washed, and resuspended with 20 mM Tris/HCl (pH
7.2). The specific Na,K-ATPase activity of the diluted treated enzyme
was 4.9 ± 0.7% of the parallel control. The data show the
K+ phosphatase activities obtained from linear time courses
of p-nitrophenol release at increasing pNPP
concentrations. To facilitate comparison, the data for the control
enzyme (open symbols) is plotted against the left-hand
axis and that for the Co(NH3)4ATP enzyme
(solid symbols) is plotted against the expanded
right-hand axis. The hyperbolae were fitted with
Vmax (µmol min 1
mg
1) and K0.5 (mM)
0.75 ± 0.03 and 4.3 ± 0.5 (control enzyme, continuous
line), and 0.09 ± 0.01 and 3.9 ± 1.1 (Co(NH3)4ATP-inactivated enzyme, dotted
line). B, effect of FITC inactivation of native sodium
pump. Parallel samples of native enzyme were incubated with or without
20 µM FITC for 4 h at 20 °C, spun down, washed,
and resuspended with 200 mM Tris/HCl (pH 7.5), 2 mM EDTA, 2 mM dithiothreitol. The specific
Na,K-ATPase activity left was 8.0 ± 0.6% of the parallel
control. The specific K+ phosphatase activities shown have
been fitted with Vmax (µmol min
1
mg
1) and K0.5 (mM)
0.96 ± 0.02 and 5.4 ± 0.3 (control enzyme, open
symbols), and 0.90 ± 0.02 and 11.4 ± 0.4 (FITC-inactivated enzyme, solid symbols), respectively.
Standard errors are contained within the symbols.
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Fig. 5.
Co(NH3)4ATP
inactivation of the K+ phosphatase activities of native and
FITC-modified Na,K-ATPase in a simultaneous experiment.
Na,K-ATPase (1.1 mg ml 1) was incubated for 2.5 h at
20 °C, pH 9.2, with or without 20 µM FITC, washed
twice by ultracentrifugation, and resuspended with 40 mM
imidazole (pH 6.8), 2 mM EDTA. The Na,K-ATPase and
K+ phosphatase activities of the FITC-enzyme were 2 and
77%, respectively, of the control enzyme activities. FITC enzyme
samples were mixed with an equal volume of water or of 1 of 5 Co(NH3)4ATP dilutions in water. After sampling
for zero time K+ phosphatase activities, the tubes (30 µl) were incubated at 37 °C for up to 80 min; six control enzyme
mixes were then started 11 min later. Four µl of each of the 12 suspensions were sampled at 0, 20, 50, and 80 min, into 1 ml of
K+ phosphatase medium containing 3-OMFP at 37 °C in a
spectrophotometer cuvette, and 3-OMF release was followed as described
under "Experimental Procedures." Top panel, control
enzyme. Samples incubated with Co(NH3)4ATP at a
final concentration of (mM) 0.15 (circles), 0.40 (reversed triangles), 0.75 (squares), 1.5 (triangles) and 3.0 (diamonds). The
K+ phosphatase activity of each sample has been
divided by the activity of the zero
Co(NH3)4ATP sample (not shown), and the natural
logarithm of the ratio has been plotted against inactivation time. The
straight lines have been fitted by linear regression to
extract the inactivation rate constant kinact
(1/slope). Middle panel, FITC-inactivated enzyme.
Co(NH3)4ATP concentrations, data treatment, and
symbols as in top panel. Bottom panel, the
kinact values obtained in the above panels have
been plotted against the Co(NH3)4ATP
concentration. Hyperbolae of the form kinact
= kinact(max)/(1 + Km/[Co(NH3)4ATP]) were
fitted with Km and
kinact(max): 0.45 ± 0.11 and 2.11 ± 0.16 h
1 (control enzyme) and 0.41 ± 0.10 mM and 1.71 ± 0.13 h
1
(FITC-enzyme).
1, respectively
(not shown). As the bulky fluorescein moiety bound to the
-chain caused less than 15% decrease in the overall effectiveness of the Co(NH3)4ATP inactivation, it would
appear as if FITC did not interfere with the binding of the ATP
analogue. However, although unlikely, the result could have arisen from
fortuitous compounding of a lower binding affinity with a higher
maximal inactivation rate in the FITC enzyme. We compared, therefore,
the Co(NH3)4ATP binding affinity and the
maximal inactivation rates of native and FITC-modified enzymes. This
was first done separately and then as a single experiment where the
FITC enzyme and its native control were incubated side by side at
various times and Co(NH3)4ATP concentrations
(Fig. 5). The design was such that the
Co(NH3)4ATP treatment of every enzyme sample
was stopped as its spectrophotometric K+ phosphatase assay
was started (3-OMFP was used as substrate). The results in Fig. 5 show
that there is not much change in K0.5 for
Co(NH3)4ATP and only a 20% reduction in the
maximal inactivation rate. We should recall that the FITC modification
inactivates E1 functions like high affinity ATP binding and
phosphorylation by [
-32P]ATP as much as the
Na,K-ATPase activity (25, 27). In this experiment, therefore,
Co(NH3)4ATP is revealing a low affinity nucleotide site in the non-cycling enzyme, one whose affinity toward
Co(NH3)4ATP seems to have experienced little
change after FITC has covalently blocked the high affinity ATP site.
protomer (27, 29, 30) or just a single site but
within the framework of a membrane-bound (
)2 dimer
whose halves work out of phase (47, 48). In the latter case, we have to
assume that only one protomer in the dimer can bind and trap
Co(NH3)4ATP, and that only this protomer loses all ATP responsiveness. One must further surmise that, through subunit
interactions, the blocked protomer should cause its neighbor in the
dimer to be confined to E1 conformations; the good protomer would not
hydrolyze ATP but could be phosphorylated by it and catalyze ATP-ADP
exchange. To test this model, we used
C12E8 to solubilize (4-6) both
Co(NH3)4ATP-modified and control Na,K-ATPase to
protomers. ATP-ADP exchange, K+ phosphatase, and
Na,K-ATPase activities and protein were determined in membrane-bound
and solubilized samples of inactivated and control enzymes. The
specific activity (in µmol min
1 mg
1) was
calculated for each reaction catalyzed by the protomers arising from
the Co(NH3)4ATP enzyme and was expressed as
percent left of the respective specific activity of the control
protomers. Equivalent calculations were made with the parent,
membrane-bound Co(NH3)4ATP-inactivated and
control samples.
)2 dimeric membrane enzyme, and as
C12E8 should have dissociated the enzyme to
protomers in our conditions (4-6), one would expect that the
good but restrained protomer should now be rid of its inactive partner.
Half of the Na,K-ATPase and K+ phosphatase activities that
had been lost would now be restored. The results in Fig.
6 show that this is not so and agree with the 2-site option; the open plus solid
bars illustrate the results expected from the single site plus
dimer hypothesis. It is also evident that the ATP-ADP exchange
activity of the Co(NH3)4ATP-treated enzyme
remains high before and after solubilization. The experiment was
repeated 12 times, with different preparations of purified enzyme and
with variable Co(NH3)4ATP exposure, so as to
obtain varying levels of inactivation; the results are shown in Fig. 7. The Na,K-ATPase and K+
phosphatase data conform rather well to the one-to-one correlation expected for two ATP sites per
protomer and are far removed from
that predicted for the single site plus dimer hypothesis (upper
dashed line). Table I presents the
analytical ultracentrifugation results that show that solubilization of
the Co(NH3)4ATP-modified enzyme with
C12E8 does actually produce a protomeric
preparation, just as it happens with the native (4-6),
FITC-treated (49), or CrATP-treated enzymes (50). There was no
difference in sedimentation coefficient between
Co(NH3)4ATP-treated and control enzyme at any
of the inactivation levels. The average
s20,w returns a protein molecular mass
of 158,000 ± 11,000, which is close to 147,500, the sum of
and
proteins (2, 4-6, 29).
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Fig. 6.
The enzymic signature of
Co(NH3)4ATP-modified Na,K-ATPase before and
after solubilization to
protomers. Paired samples of native enzyme were incubated
for 2.5 h at 37 °C in 20 mM imidazole (pH 6.3) with
or without 1 mM Co(NH3)4ATP. The
samples were centrifuged, washed, and resuspended with 300 mM NaCl, 40 mM TES (pH 7.4), and their protein
concentrations were determined and adjusted to 680 µg
ml
1. The Na,K-ATPase activity was assayed in 1:20 enzyme
dilutions of the control and
Co(NH3)4ATP-modified enzymes, as described
under "Experimental Procedures" (except that 10 mM TES
(pH 7.5) replaced the imidazole); the K+ phosphatase and
ATP-ADP exchange activities were determined likewise (with 10 mM TES, pH 7.5, and using 20 mM pNPP
for the former and 150 mM NaCl for the latter). The
specific activities of the modified membrane-bound enzyme
(MB, cross-hatched bars) are shown as percent of
the respective specific activities of the control enzyme. Aliquots of
the modified and control enzymes were diluted 1:1 with
C12E8 (2 mg ml
1) to
obtain soluble
protomers, and protein and enzymic activities
were now determined in the 425,000 × g supernatants.
The specific activities of the protomeric
Co(NH3)4ATP-enzyme are shown
(
, solid bars) as percent of the specific
activities of the control protomeric enzyme. The open plus
solid bars represent percent specific activities that
would be expected for the soluble protomer if the membrane-bound enzyme
consisted of (
)2 dimers with a single ATP site per
protomeric unit. Vertical lines are compounded errors
of the mean.
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Fig. 7.
Correlation between the Na,K-ATPase and
K+ phosphatase activities of
Co(NH3)4ATP-modified, membrane-bound sodium
pump and the respective activities of the resulting soluble
protomers. Paired samples of membrane-bound Na,K-ATPase were
incubated with and without Co(NH3)4ATP as in
Fig. 6, in different experiments, so as to obtain various degrees of
inactivation. The protein concentrations and enzymic activities were
determined in the Co(NH3)4ATP-modified enzyme
and in the parallel control before and after solubilization to
protomers. The specific activities of soluble and (parent)
membrane-bound inactivated enzymes have been expressed as in Fig. 6 and
plotted against each other. Solid symbols, Na,K-ATPase
activity; open symbols, K+ phosphatase activity.
Bars show the compounded errors. Dashed and
dotted lines represent expectations for the hypotheses
tested; for explanation, see main text.
Analytical ultracentrifugation of
Co(NH3)4ATP-modified Na,K-ATPase and
native Na,K-ATPase after solubilization with C12E8
1(4), the calculated protein molecular mass
works out as 158,000 ± 11,000. The specific Na,K-ATPase
activities were determined in samples of the inactivated and parallel
control enzymes; the former is shown as percent of the latter.
Protomers--
Because of the
limited thermal stability of the soluble enzyme (49), we first tried
lowering the temperature to 20 °C to demonstrate direct
Co(NH3)4ATP inactivation of the soluble
protomers over periods similar to those in Fig. 2. As this was
unsuccessful, we decided to take advantage of the greater thermal
stability of the soluble
protomer at 37 °C when in the
presence of low K+ concentrations, in Na+-free,
Mg2+-free medium (49). Fig.
8A (open circles)
shows that the soluble protomers were quite stable at 37 °C in the
presence of 10 mM K+. This panel also shows
that 50 µM Co(NH3)4ATP was
exceedingly effective under these conditions, causing enzyme
inactivation at a rate much faster than obtained with the
membrane-bound enzyme at higher analogue concentrations (compare with
Fig. 2). When ATP was included in the medium, the inactivation rate
decreased to less than half. The experiment in Fig. 8B shows
the Co(NH3)4ATP concentration dependence of the
inactivation process. In this case, the rate constant
kinact has been estimated from a single time
point (2.5 min) under the assumption of single exponential decay, and
this can only be an approximation. It seems unlikely, however, that
this degree of uncertainty could explain the marked differences with
respect to the kinetics of inactivation of the membrane-bound enzyme
(Fig. 3A). Relative to the latter, the maximal inactivation
rate of the soluble protomers was now 12 times faster, and the affinity
nearly 40-fold higher during the Co(NH3)4ATP binding event that precedes irreversible enzyme modification.
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Fig. 8.
Direct Co(NH3)4ATP
inactivation of soluble protomeric Na,K-ATPase. The native enzyme
was solubilized with C12E8 in a medium
containing 10 mM KCl, 20 mM TES (to pH 7.25 with triethanolamine). The K+ phosphatase activity was
measured in 1:6 dilutions of the high speed supernatant, as linear time
courses over 8 min at 20 °C. A, soluble protomers were
incubated at 37 °C; samples were stopped by dilution and cooling at
the times shown, and their K+ phosphatase activity was
determined. Three separate runs, with (i) no additions (open
circles), (ii) 50 µM
Co(NH3)4ATP (solid circles), and
(iii) 50 µM Co(NH3)4ATP plus 2 mM ATP (open triangles) or without ATP
(solid triangles). Vertical lines show the S.E.
B, soluble protomers were preincubated for 2.5 min at
37 °C, at the Co(NH3)4ATP shown, and their
K+ phosphatase activity was determined as above. An
estimate of the inactivation rate constant
kinact has been plotted on the
ordinate; the data have been fitted to the hyperbola shown,
with KD = 16 ± 1 µM and
kinact(max) = 28.8 ± 0.6 h 1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protomer, to explain the unfaltering
Co(NH3)4ATP inactivation of the FITC-modified membrane enzyme (Fig. 5), we would need to postulate that the minimal
functional unit of Na,K-ATPase was an (
)2 membrane
dimer, at the very least. The Co(NH3)4ATP
inactivation pattern observed with the native membrane-bound
enzyme could then be explained if the analogue was trapped by only
one-half of the hypothetical dimer. This would lock the other half in
an E1 conformation (32) and allow substantial survival of the ATP-ADP
exchange activity while causing total loss of Na,K-ATPase activity. We
applied the solubilization test used earlier (29) which showed that
FITC could access and inactivate the high affinity ATP site of every
protomer in the membrane-bound enzyme. In the present case, the
expectation with respect to the
Co(NH3)4ATP-modified enzyme was that
solubilization to single protomers would increase the Na,K-ATPase
activity if the putative membrane-bound (
)2 dimer had
one good and one Co(NH3)4ATP-blocked protomer,
with one ATP site each protomer. The experiments in Figs. 6 and 7 show
that this was not the case. At face value, the conclusion must that both ATP sites should be found on every
protomer.
protomer. Table I shows that the sedimentation coefficients obtained for the
Co(NH3)4ATP-modified and native enzymes were
identical and returned, within error, the protein molecular mass of the
particle (4-6). By applying the method of active enzyme
ultracentrifugation, we had shown earlier (6) that in our
solubilization conditions (weight ratio
C12E8/protein = 3) the
protomer was
the only soluble species with Na,K-ATPase activity. We then also found
that the dual nucleotide effects on the activity were inherent to the
soluble protomeric enzyme (6), i.e. there was no need to
advocate a membrane (
)2 dimer to explain this basic
feature. However, our results are still at odds with those obtained by
high performance gel filtration/laser light scattering of the
C12E8-solubilized enzyme (52, 53), which shows
a substantial level of (
)2 dimers in equilibrium with
the
protomer. Although we do not have an explanation for the
discrepancy, we have pointed out (6, 29) that, in the event of such
equilibrium, our s20,w measurements
always return an upper estimate of the weighted-average molecular mass of protomer and putative dimer (54), because of dimer dissociation upon
dilution. The steady-state properties of the gel filtration protomers
and dimers have not been assessed (52, 53) but, even using
KA = 5 × 105
M
1 at 20 °C (52), at a protein
concentration
1.9 × 10
7 M (Figs.
6-8), the calculated contents of soluble dimers works out as
<7%.
protomer in the (
)2 membrane dimer is
blocked directly by Co(NH3)4ATP (or FITC for
that matter), total loss of Na,K-ATPase activity can only result from
restrictive subunit interactions within the dimer. But then as protomer
and dimer will be in rapid equilibrium after solubilization, one can
safely predict that good and bad
protomers will be instantly
randomized within the small soluble (
)2 population.
For instance, if the E2 partial reactions and the Na,K-ATPase activity
of the membrane enzyme are 100% inactivated, the outcome of
solubilization/recombination will be 50% hybrid dimers, 25%
twice-inactivated dimers, and 25% intact dimers, i.e. the
reshuffle shall give away the presence of the stealthy dimer. Let
p be the probability of intact
solubilized from
membrane (
)2 at each inactivation level,
i.e. p = 0.5 at zero membrane activity, 0.75 at 50% membrane activity, etc. The probability of forming active
soluble dimers is then p2 which, expressed as
percent, is shown by the dotted line in Fig. 7. As only the
good soluble (
)2 shall be active, the relative specific activity of the soluble enzyme must be equal to the ratio (good soluble dimer concentration per mg of inactivated enzyme)/(good soluble dimer concentration per mg of control enzyme). From the basic
expression for a protomer/dimer equilibrium it is easily shown that, at
low dimer levels (i.e. the condition for the "concealed dimer" proposal (48)), the above ratio is very approximately p2. This is because we should expect a unique
association constant KA for the dimerization of
intact soluble
, whether it arises from the inactivated or the
control enzyme. Therefore, the dotted line in Fig. 7 also
represents the predicted correlation for the relative specific activity
of the soluble enzyme, expressed as percent. That was not the
correlation for either Co(NH3)4ATP (Fig. 7) or
FITC (Fig. 3 in Ref. 29). The option of an active soluble dimer in fast
equilibrium with an inactive protomer (48) may, therefore, be discarded.
Protomer--
By incubating at
pH 7.25 and 37 °C, we could demonstrate
Co(NH3)4ATP-dependent inactivation
of more than 90% of the solubilized enzyme (Fig. 8A),
despite the presence of K+ (31). This shows that all
protomers are intrinsically and equally susceptible to the
analogue and reinforces the results of Figs. 6 and 7. An interesting
feature is the ~40-fold increase in
Co(NH3)4ATP binding affinity,
KD = 16 µM (Fig. 8B), by
comparison with the membrane-bound enzyme, KD = 0.41-0.62 mM (Figs. 3A and 5C). This
lower KD is as would be expected from the ATP
concentration dependence of the Na,K-ATPase activity of the soluble
protomer at 20 °C (6), where the low affinity K0.5 was 21 µM (as opposed to 175 µM for the membrane-bound enzyme). The maximal turnover
rates of membrane-bound and solubilized enzymes are very similar, in
conditions such that low affinity ATP binding controls the
rate-limiting step (4-6). This suggests that the lower
K0.5 reflects a decrease in real dissociation
constant (KD = koff/kon) at the low
affinity site. It is perhaps a lower koff that
better explains this change, as a 10-fold faster
Co(NH3)4ATP inactivation rate constant
(kinact) was observed with the soluble protomer
(Figs. 3A and 8B). A slower off-rate will result
in a longer lifetime for the initial
[enzyme·Co(NH3)4ATP] complex in detergent
solution; the complex should thus have the opportunity to sample a
greater number of sub-conformational states, with a higher probability
of finding that conducive to Co(NH3)4ATP trapping and site inactivation. Of course, the higher ATP and Co(NH3)4ATP affinities in
C12E8 solution may be the result of changes in
protein conformation caused by detergent binding and the formation of
protein micelles. It is possible that the phase transitions
independently increase the rate of Co(NH3)4ATP
inactivation as well.
1-Na,K-ATPase, by assimilation to the N domain of SERCA (24).
Mutants were expressed in HeLa cells and selected by virtue of their
ouabain resistance; none of the mutations drastically affected low
affinity ATP binding. The difficulty with this approach is that the
mutants sought could not have been viable, because their
Na+ pumping rate would have been less than 5% normal.
Because the endogenous pump was inhibited by ouabain, the rise of
intracellular Na+ would have caused colloid-osmotic
swelling and lysis of the host cells. Despite the selection procedure,
it could be found that in the case of R544A there was a 59-fold
increase in K0.5 for phosphorylation by ATP and
only a 2-fold increase in K0.5 for the low
affinity ATP effect (57). This shows that it is at least possible to
interfere with high affinity ATP binding with little effect on low
affinity binding.
-methylene]triphosphate (59) inactivates ATP
phosphorylation with a high affinity but does not affect E2 reactions,
i.e. it has effects opposite to those of
Co(NH3)4ATP. Among agents that bind covalently
to Na,K-ATPase, p-fluorosulfonylbenzoyl-adenosine (60),
8N3-ATP (27), 2N3-ATP (61), and FITC (25, 29)
are, at low concentrations, far more effective at inactivating the
Na,K-ATPase than the K+ phosphatase activity. These
affinity labels become incorporated in the
subunit at
Lys719, Lys480, Gly502, and
Lys501, respectively (22, 62-64).
-32P]ADP photolabels
Lys480 of native Na,K-ATPase, i.e. the same
residue as 8N3-ATP (63); the equivalent Lys492
is the TNP-8N3-ATP target in the N domain of SERCA (71).
But then close to 1 mol of
TNP-8N3-[
-32P]ADP can be incorporated per
mol of FITC-modified
Na,K-ATPase, with a lower affinity (30), now in
the tryptic hexadecapeptide beginning at
Ala721 of the P
domain (69, 70). In the open SERCA structure (24), Lys492
is 25-30 Å away from the fragment equivalent to
Ala721-Lys736 in Na,K-ATPase; this distance
is similar to that between FITC and Co(NH3)4ATP
in the sodium pump (65, 66). The P domain might then be one of the
platforms accommodating a low affinity ATP-binding site. This might
also be the case with the site for the phosphatase substrate, as
4N3-2-nitrophenyl phosphate can photolabel Na,K-ATPase at
Pro668 and
Asn398 (72), presumably in its
P and N domains, respectively.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. W. Schoner (Justus-Liebig Universität, Giessen, Germany) for samples of Co(NH3)4ATP and advice on its synthesis and use, and Prof. A. Rowe, National Centre for Macromolecular Hydrodynamics, for help with the analytical ultracentrifugation.
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FOOTNOTES |
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* This work was supported by research grants from The Wellcome Trust and the Medical Research Council.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.
Present address: School of Biosciences, University of Birmingham,
Edgbaston, Birmingham B15 2TT, UK.
§ To whom correspondence should be addressed. Tel./Fax: 44-116- 2523091; E-mail: jdc7@le.ac.uk.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211128200
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ABBREVIATIONS |
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The abbreviations used are: Na, K-ATPase, (Na+ + K+)-activated adenosine triphosphatase; Co(NH3)4ATP, Co(III) tetrammine ATP; Co(NH3)4PO4, Co(III) tetrammine phosphate; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; C12E8, octaethylene glycol dodecyl monoether; pNPP, p-nitrophenyl phosphate; 3-OMFP, 3-O-methylfluorescein phosphate; 3-OMF, 3-O-methylfluorescein; FITC, fluorescein 5'-isothiocyanate; TNP-AMP, -ADP, or -ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5' mono-, di-, or triphosphate; 2- or 8N3-ATP, 2- or 8-azido-adenosine 5'-triphosphate; TNP-8N3-ADP, 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-diphosphate; ErITC, erythrosin 5'-isothiocyanate; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; HPLC, high pressure liquid chromatography.
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REFERENCES |
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