From the ¶ Department of Biochemistry and Molecular Biology,
Oregon Health and Science University, Portland, Oregon 97201, the
Department of Biological Sciences, Illinois State
University, Normal, Illinois 61790, and the § Department
of Biological Sciences, Eastern Illinois University,
Charleston, Illinois 61920
Received for publication, December 4, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The reaction mechanism of the Na,K-ATPase is
thought to involve a number of ligand-induced conformational changes.
The specific amino acid residues responsible for binding many of the
important ligands have been identified; however, details of the
specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify
interactions between domains of the Na,K-ATPase The Na,K-ATPase is an integral membrane protein that plays a
central role in ionic homeostasis in animals by mediating the translocation of Na+ and K+ ions against their
electrochemical gradients across the plasma membrane (for a review, see
Ref. 1). The Na,K-ATPase functions as a heterodimer composed of a
105-kDa Recently, the three-dimensional structures of several
P2-type ATPases including the H-ATPase (6), the Na,K-ATPase
(7), and the sarcoplasmic reticulum Ca-ATPase (8, 9) at 8-, 11-, and
2.6-Å resolution, respectively, have been solved. As one might predict, the high resolution sarcoplasmic reticulum Ca-ATPase (SERCA)1 pump structure shows
that the 10-transmembrane segments are largely The Na,K-ATPase and the gastric H,K-ATPase are the only members of the
P2-type ATPase family that possess two obligatory
subunits, The oligomeric state of the Na,K-ATPase remains a controversial issue.
Indeed, it seems hard to dispute the findings that monomeric
(i.e. detergent-solubilized) In this paper, we demonstrate that the isolated nucleotide binding
domain can directly associate with full-length Na,K-ATPase purified
from dog kidney. In addition, this interaction was significantly enhanced by the binding of magnesium and ATP. To further investigate the sites of interaction, we determined whether the isolated
ATP-binding domain (ABD) could interact with itself in the absence of
other pump domains. In the presence of MgATP, we found that a
GST-tagged ABD associated with a His6-tagged ABD.
Consistent with the facilitation of interaction by nucleotide binding
was the observation that both FITC and eosin significantly decreased
the degree of association. Taken together, these data imply that the
Na,K-ATPase is capable of self-association and that this quaternary
structure is stabilized upon MgATP binding. Preliminary accounts of
this work have been previously reported (25).
Reagents and Media
Glutathione-Sepharose 4B and Rainbow protein molecular weight
markers were from Amersham Biosciences. NaCl, KCl,
MgCl2, Na2HPO4, NaH2PO4, glutathione, Tris-base, Coomassie
Brilliant Blue R-250, phenylmethylsulfonyl fluoride, antipain,
leupeptin, pepstatin A, FITC, eosin, and imidazole were purchased from
Sigma. Recombinant streptavidin-tagged superoxide dismutase was a
generous gift of John Eisses (Oregon Health and Science University).
The bacterial lysis reagent, BPER II, was purchased from Pierce.
The primary antibody, mouse anti-penta-His, was obtained from Qiagen,
and the secondary antibody, horseradish peroxidase-conjugated goat anti-mouse, was from Sigma.
Protein Expression and Purification
Escherichia coli was used to overexpress the M4M5
cytoplasmic loop from GST-M4M5 Loop Purification
The GST construct was produced via PCR amplification of a
1260-bp section encoding the M4M5 loop from a pGEM-rat A single colony was grown overnight and used to inoculate 500 ml of
LBamp (100 µg/ml) containing 2% ethanol. (We previously observed that the presence of ethanol dramatically increased the amount
of fusion protein appearing in the soluble fraction after bacterial
cell lysis (26).) After an A600 of 0.8 was
attained, protein synthesis was induced with 0.1 mg/ml isopropyl
His6-M4M5 Loop Purification
The His6-tagged M4M5 loop was produced and purified
as described previously (26). Briefly, the identical region of the rat Binding of C12E8-solubilized Dog Kidney
Na,K-ATPase to Immobilized Fusion Protein ATP-binding Domains
Dog kidney Na,K-ATPase was purified by the method of Jorgensen
(29) with the modifications described previously (30). In order to
assay for binding between the immobilized GST-ABD and dog kidney
ATPase, 50 µg of dog kidney enzyme in 200 µl of 50 mM
Tris (pH 7.4) and 0.1% C12E8 was combined with
a 200-µl slurry of GST-ABD conjugated to glutathione-Sepharose in the
presence or absence of enzyme substrates (as shown in the figure
legends). Control assays were performed using slurries of
glutathione-Sepharose 4B that was not bound to GST-ABD. Microcentrifuge
tubes containing the 400-µl reaction slurries were rotated at room
temperature for 60 min, and the unbound enzyme was removed by
centrifugation (1000 rpm in a tabletop microcentrifuge for 5 min). The
supernatant was discarded, and the Sepharose pellet was resuspended in
1 ml of 50 mM Tris (pH 7.4, with corresponding substrate
where appropriate) and rotated at room temperature for 5 min. The
supernatant was removed, and the Sepharose was washed two additional
times in the same manner.
Proteins were removed from the Sepharose by the addition of 100 µl of
Laemmli sample buffer (i.e. 1:1:1 (v/v/v) of 8 M
urea, 10% SDS, and 125 mM Tris-HCl, pH 6.8, and 5%
Interaction between Expressed ATP-binding Domains via
"Pull-down" Assays
The GST-ABD fusion protein was bound to the
glutathione-Sepharose and subsequently washed with TBS (50 mM Tris, 120 mM NaCl, pH 7.4) to remove unbound
fusion protein. Domain-domain interactions were initiated by adding 50 µg of the His6-ABD to a slurry of the conjugated GST-ABD
in a final volume of 200-400 µl of TBS alone or containing various
ligands (see figure legends for details). The fusion proteins were
rotated at 4 °C for 1-3 h. After the interaction period, the
glutathione-Sepharose was pelleted via centrifugation (1000 rpm in a
tabletop microcentrifuge for 5 min) and washed three times with a
20-volume quantity of 50 mM TBS, containing any additives
that were present during the interaction period. Finally, the GST loop
and any His6 loop bound to it were eluted from the
Sepharose with 100 µl of 10 mM reduced glutathione or 100 µl of Laemmli sample buffer. Proteins were separated via SDS-PAGE and
electrotransferred to PVDF as described above. Evidence for
His6-ABD interaction with GST-ABD was demonstrated via
immunostaining with mouse anti-penta-His antibody (Qiagen) and
horseradish peroxidase-conjugated goat anti-mouse IgG secondary
antibody (Sigma).
Treatment of Fusion Protein ABDs with ATP Site Probes
Eosin--
Eosin, tetrabromofluorescein, has been shown to be a
potent inhibitor of the Na,K-ATPase by competing with ATP (32). Thus, we performed the domain-domain interaction experiments described above
in the presence and absence of 10 µM eosin.
FITC--
FITC is a potent irreversible inhibitor of the
Na,K-ATPase and has been shown to specifically label
Lys501, which resides in the nucleotide binding site (33,
34). The GST loop was modified by incubating the protein, bound to
glutathione-Sepharose 4B, with 20 µM FITC in 50 mM Tris buffer (pH 9.0, 30 min, 25 °C). The unbound FITC
was removed by washing the Sepharose twice with a 10-volume quantity of
TBS. FITC labeling was confirmed by UV illumination of the labeled
protein on a gel (see Fig. 7B, middle panel). The His6 loop was labeled similarly,
except the protein was not bound to the
Ni2+-nitrilotriacetic acid; thus, excess FITC was removed
via dialysis (12-kDa cut-off) overnight against a 1000-volume quantity
of 50 mM Tris (pH 8.0). In order to determine whether FITC
modification was necessary at either or both ABD partners, both
FITC-labeled and unlabeled GST-ABD were incubated in the presence of 5 mM MgATP and 50 µg of either FITC-labeled or unlabeled
His6-ABD.
Na,K-ATPase Activity Measurements--
Na,K-ATPase activity was
determined in a standard assay medium containing 1 mM EGTA,
130 mM NaCl, 20 mM KCl, 3 mM
MgCl2, 3 mM Na2ATP, 50 mM imidazole, pH 7.2, and 0.5 µg of purified dog kidney
enzyme (or enzyme solubilized with C12E8). The
mixture was incubated at 37 °C for 15 min, and the amount of
inorganic phosphate released through ouabain-sensitive ATP hydrolysis
was measured as described previously (35).
Phosphorylation with [32P]ATP--
The
phosphorylation measurements were carried out essentially as described
previously (36) in 50 µl of medium containing 100 mM NaCl
(or 100 mM KCl), 5 mM MgCl2, 50 mM Tris-HCl, pH 7.2, and 50 µg of protein. The reaction
was initiated by the addition of ATP ([ Interaction between Purified Dog Kidney Na,K-ATPase with a
Recombinant ATP-binding Domain--
For these studies, a GST fusion
protein of the large cytoplasmic loop between the fourth and fifth
transmembrane segments was constructed. We reported previously that
this GST-M4M5 loop was able to bind ATP as determined via protection
against FITC labeling (37). More recently, the corresponding domain in
the full-length rabbit fast twitch SERCA has been shown to
coordinate TNP-AMP binding by x-ray crystallographic analysis (8).
To determine whether the GST-M4M5 loop (GST-ABD) associated with
purified full-length Na,K-ATPase,
C12E8-solubilized dog kidney Na,K-ATPase (50 µg) was incubated at 25 °C with a slurry of the GST loop bound to
a glutathione-Sepharose affinity resin. The incubation was performed
both in the absence and presence of pump substrates (Fig.
1A). There was a clear
interaction between the GST-ABD and the intact Na,K-ATPase in the
presence of MgATP (Fig. 1A, lane 6).
Occasionally, we observed that there was a less prominent band in the
presence of MgADP as well (Fig. 1A, lane
5), suggesting that MgADP may also promote an interacting
conformation. Specifically, we observed a MgADP-facilitated interaction
in three of five experiments where the PVDF membrane was probed with an
anti-
We next determined the concentration of MgATP required to stabilize the
interaction between the GST-ABD and
C12E8-solubilized sodium pump. These
experiments are possible because the affinity for MgATP binding to the
full-length Na,K-ATPase is more than 3 orders of magnitude greater than
to the bacterially produced constructs (see "Discussion") (26).
Fig. 2 shows that near millimolar MgATP
concentrations are necessary to stabilize the heterodimer between
GST-ABD and the full-length pump.
Evidence Suggesting That Interactions Take Place within the
Nucleotide-binding Domain--
The Na,K-ATPase nucleotide-binding
domain comprises nearly 40% of the mass of the catalytic
However, the segment (or segments) of the full-length Na,K-ATPase that
interacted with the bacterially produced ATP-binding domains remained
to be determined. Thus, we initially designed experiments to determine
whether the interactions occurred between the two ATP-binding domains
(i.e. the M4M5 loop of the intact enzyme with the equivalent
heterologously expressed isolated domain). We tested this hypothesis by
measuring interactions between the two purified cytoplasmic loops
themselves. The soluble His6-ABD was incubated with GST-ABD
bound to glutathione-Sepharose resin in the presence of varying
substrates. We observed that the two constructs did associate and that
this association depended upon the simultaneous presence of both
magnesium and ATP (Fig. 4A). In other words, neither ATP alone nor magnesium alone was an effective promoter of the interaction. MgADP and MgAMP were unable to facilitate interaction between the two nucleotide-binding domains (Fig.
4B). The inability of MgADP to promote interactions between
the GST-ABD and the His6-ABD, was somewhat surprising,
considering that it did facilitate interactions between the GST-ABD and
intact Na,K-ATPase (Fig. 1A). Thus, this finding may suggest
that MgADP binding to the full-length
It was important to ensure that MgATP does not facilitate nonspecific
protein interactions with GST-ABD or glutathione-Sepharose. Therefore,
we incubated a bacterially produced superoxide dismutase (SOD) fusion
protein with the GST-ABD under the same conditions that promote
interaction with the His6-ABD. Fig.
5 shows that there was no interaction
between the GST-ABD and SOD in the presence or absence of MgATP. The
Western blot was probed with an anti-SOD antibody, and a positive
control lane shows that the immunostaining was successful (Fig. 5) and
that SOD would have been detectable had it interacted with the
GST-ABD.
Specificity of the Interactions between ATP-binding
Domains--
To more directly examine the MgATP-dependent
association between these ATP-binding domains, we determined whether
known inhibitors of ATP binding to the Na,K-ATPase could block the
interaction. For example, eosin is a reversible inhibitor of the
Na,K-ATPase, and the binding of eosin and ATP are mutually exclusive
(32). Thus, eosin should block GST-ABD and His6-ABD
interactions, by preventing MgATP binding. Consistent with this
prediction, the presence of 10 µM eosin greatly reduced
the binding of His6-tagged loop to GST-tagged loop in the
presence of 1 mM MgATP (Fig.
6). Both the Coomassie Blue-stained gel
(Fig. 6A) and the anti-penta-His stained Western blot (Fig.
6B) indicate that a much smaller amount of the
His6-tagged loop bound to the GST fusion protein in the presence of eosin.
FITC is a fluorescent amine-reactive molecule that labels
Lys501 in the purified Na,K-ATPase; this reaction is
prevented by the simultaneous presence of ATP (33, 34). Similarly, ATP
has been shown to protect both the His6-ABD (26) and the
GST-ABD (37) against FITC labeling. When both the GST-ABD and
His6-ABD were labeled with FITC, domain-domain interactions
were substantially reduced (Fig.
7B, right
panel). Fig. 7B (middle
panel) shows FITC incorporation into the GST-ABD, whereas
FITC-labeled His6-ABD is not observed, since it did not
associate with the GST-ABD and thus was lost during the washing steps.
(The FITC labeling protocol used for His6-ABD modification
was identical to those published previously (26).) Interestingly, FITC
labeling of only one of the interacting fusion proteins did not inhibit
the interaction; clearly, FITC modification of the His6-ABD
(Fig. 7A, middle panel) did not
significantly reduce its ability to associate with the nonmodified
GST-ABD (Fig. 7A, right panel).
Phosphorylation Cannot Explain Domain-Domain Interactions--
It
is clear that MgATP, and not ATP alone, promotes the observed ABD
interactions. An obvious possibility might be that the proteins are
undergoing magnesium-dependent phosphorylation. However, there was no difference between the K+-containing control
with the intact Na,K-ATPase and either the His6-ABD or the
GST-ABD in the presence of Na+ or K+ (Fig.
8). This may not be surprising, since the
isolated ABDs are devoid of cation-binding sites. Nonetheless, the
phosphorylation levels observed for the fusion proteins (Fig. 8),
compared with the level of 32Pi captured on the
filter with perchloric acid-denatured protein (i.e.
background), was minimal. Moreover, considering that the fusion
proteins are less than half the molecular mass of the Na,K-ATPase and
constitute a purer protein preparation, the difference in phosphoprotein production between the intact enzyme and the fusion proteins is a very conservative estimate (Fig. 8). Also, considering that neither the His6-ABD nor the GST-ABD can hydrolyze ATP
(data not shown), the inability to isolate a phosphorylated ABD (Fig. 8) cannot be due to a more labile acyl-phosphate intermediate. Rather,
it seems likely that the ABD constructs cannot "close" sufficiently
to bring the N and P domains together, a requirement of phosphoenzyme
formation (8, 9). This inability to close is probably due to the
lack of both the membrane domains and the A domain in the
isolated ABDs. Thus, our data suggest that the protein conformation
producing the strongest domain-domain interactions is produced simply
by the binding of both magnesium and ATP and not phosphoenzyme
formation.
The high resolution structures of the Ca-ATPase (8, 9) clearly
show that, as predicted previously (41), the cation and nucleotide
binding domains of P2-type ATPases are not only functionally separate but are spatially separate as well. In other words, all of the residues involved in ATP-binding and hydrolysis are
located in the cytoplasmic loop between M4 and M5, forming the two
separate N and P subdomains. Conversely, all of the residues suggested
to be involved with cation coordination in the occluded state are
located in the transmembrane-spanning regions of the enzyme (1). Since
occupation of the cation-binding site dramatically alters nucleotide
affinity, it is obvious that communication exists between the
cation-binding membrane domains and the cytoplasmic ATP-binding domain.
Indeed, the conformational changes observed between the E1
and E2 SERCA structures (see Refs. 8 and 9, respectively) show dramatic movements of transmembrane helices and changes in their structure, which push and pull on large
cytoplasmic domains. Instability of helices associated with cation
binding has been observed previously in both the Na,K-ATPase (10) and the gastric H,K-ATPase (11).
In the current work, we provide evidence that tight protein-protein
interactions occur between two differentially tagged constructs of the
large cytoplasmic loop between M4 and M5 of the Na,K-ATPase. This loop
has been shown to contain all of the residues that compose the
nucleotide binding domain in the sodium pump (26) as well as other
members of the P2-type ATPase family (8, 42). The ABD-ABD
interaction reported here was dependent upon the presence of both
magnesium ions and ATP, yet neither fusion protein was phosphorylated
(Fig. 8); nor did they possess significant ATPase activity (26).
One Versus Two ATP Molecules Bound--
Whether the
MgATP-dependent interaction between the M4M5 loops required
both partners to have MgATP bound remained a question. Thus, we
determined the MgATP concentration dependence for the interaction
between GST-ABD and intact (C12E8-solubilized)
Na,K-ATPase. It was shown previously that the isolated ATP-binding
domain has a Kd(ATP) of ~500
µM, consistent with the ABD existing in an
E2-like conformation (26). In contrast, the binding
affinity for ATP of intact Na,K-ATPase is less than 1 µM
(43). Thus, we measured interactions between the GST-ABD and intact
enzyme at varying MgATP concentrations (Fig. 2). At low MgATP
concentrations (e.g. <50 µM), where intact
enzyme is saturated with ATP and GST-ABD is less than 10% occupied by
ATP, no protein-protein interactions were detected (Fig. 2). However,
at MgATP concentrations greater than 500 µM, when both
the GST-ABD and intact enzyme are largely in the ATP-bound state, the
GST-ABD was able to pull down the intact Na,K-ATPase
If ATP-binding domain interactions only occur when each protein is in
the nucleotide-bound form, then modification of one of the fusion
proteins with FITC should eliminate ABD-ABD interactions, because the
FITC-labeled partner would be unable to bind MgATP. However, we found
that labeling either the GST-ABD or the His6-ABD could
not disrupt the MgATP-dependent association
between the two (Fig. 7A). Rather, FITC modification of both
fusion proteins was required to block the GST-ABD/His6-ABD
interaction (Fig. 7B, right panel).
Consequently, it appears that MgATP binding is only necessary to one of
the constructs to facilitate protein-protein interactions, or that a
single ATP molecule is binding in part to both fusion proteins and
forming a bridge between the two ABDs. In other words, FITC modifies
Lys501 that forms part of the binding site for the
adenosine moiety in the N domain of the Na,K-ATPase Intermolecular Interactions between Adjacent Sodium
Pumps--
Whether P-type ATPases exist as monomers, dimers
(diprotomers for the H,K- and Na,K-ATPases), or higher oligomers is a
subject of debate. There have been several reports demonstrating
oligomeric forms of various P-type ATPases (e.g. the
sarcoplasmic reticulum Ca-ATPase (44), the Na,K-ATPase (45), and the
H,K-ATPase (46)). In particular, there have been several studies that
suggest the Na,K-ATPase exists as an
It has been suggested that the findings of the
cross-linking and energy transfer studies could be a result of
the high density of Na,K-ATPase in kidney membrane preparations used in
these studies (19). Indeed, a recent study reported that thermal
denaturation of the Na,K-ATPase resulted in the formation of
Blanco et al. (49) demonstrated associations between
different Significance of A Cell Biological Role of Dimerization?--
Recently, Caplan and
colleagues (53) initially identified, using the yeast two-hybrid
system, several cellular proteins that interacted with the Na,K-ATPase.
Following careful characterization, specific candidates were
identified: 1) polycystin-1, involved in polycystic kidney disease; 2)
SNAPAP, a protein involved in vesicular targeting; and 3) the catalytic
subunit of protein phosphatase 2a (53). Interestingly, the site of
interaction of the Na,K-ATPase with these proteins was suggested to be
the M4M5 loop by measuring direct protein-protein interactions with a
bacterially purified GST-M4M5 loop, a preparation equivalent to the
construct used in our
studies.2 Furthermore, the
interactions of these foreign proteins with the GST-M4M5 loop were
significantly enhanced by the presence of MgATP.2
Considering the observations of Caplan and co-workers in light of our
MgATP-facilitated ABD-ABD dimerization, it is possible that the role of
sodium pump dimerization may be related to proper trafficking or
regulation (or both) via protein-protein interactions. In other words,
the association of other cellular regulatory proteins (e.g.
protein phosphatase 2a or SNAPAP) may depend upon the pump first
self-associating and then forming a binding site for the regulatory partner.
ABD-ABD Dimerization Mimicking Intramolecular
Interactions--
The E1Ca SERCA structure (8) shows that
the M4M5 loop is separated into two distinct structures comprising the
nucleotide-binding domain (N domain) and the phosphorylation domain (P
domain). These two domains are separated by ~50 Å in E1;
thus, it was predicted that a significant conformational closure must
occur for the transfer of phosphate from the nucleotide (in the N
domain) to the catalytic aspartate residue (in the P domain). This
closure was observed recently with the thapsigargin-stabilized high
resolution E2Tg SERCA structure (9). Although these two
structures have been invaluable for understanding the
structure-function relationship of the P-type ATPases, the steps that
occur between E1Ca and E2Tg remain unclear. It
appears that the binding of MgATP to the N domain results in a
conformational change that is more receptive for interacting with the P
domain. In addition, significant concomitant changes within the
cation-binding domain in the membrane along with the A domain also
occur between E1Ca and E2Tg (9). However, what
remains unresolved is whether initial movements between the N and P
domains produce changes in the cation-binding and A domains, or
vice versa.
In the intact Na,K-ATPase, closure of the N and P domains occurs in a
single
An alternative explanation is that MgATP forms a chemical bridge
between the GST-ABD and the His6-ABD. In this case, the
adenine moiety could bind to a portion of the N domain on one
construct, and the phosphate chain would occupy the appropriate
contacts on the adjacent construct. In the experiments where only one
construct was FITC-modified, that fusion protein would provide the
phosphate chain contacts, whereas the unmodified construct could
coordinate the adenine moiety. In this case, MgADP may be too short to
effectively bridge the two constructs together.
Conclusions--
We have shown that two separate constructs of
Na,K-ATPase ATP-binding domain can interact with each other or with
C12E8-solubilized intact enzyme. Moreover, this
interaction is greatly enhanced by MgATP binding. Although our data
cannot rule out the possibility of higher oligomeric structures, the
simplest explanation for these interactions is that the sodium pump can
exist as a dimer under certain circumstances. Indeed,
ATP-dependent dimerization of the two ATP-binding domains
within MJ0796 (an ABC transporter), has been demonstrated recently
(54). The ATP-mediated ABD dimerization in the ABC transporters has
been suggested to be critical for coupling ATP hydrolysis to substrate
translocation (55).
Alternatively, such dimerization between Na,K-ATPase ABDs may perform a
necessary cellular function as a membrane anchor for other signaling
proteins (24, 53). Experiments are under way to confirm whether the
associations presented here are dimers or higher oligomers and to
further understand the role that these sodium pump interactions may
play in cellular physiology.
-subunit that depend
on the presence of particular ligands. The major cytoplasmic loop
(between TM4 and TM5), which we have previously shown contains the
ATP-binding domain, was overexpressed in bacteria either with a
His6 tag or as a fusion protein with glutathione
S-transferase. We have observed that these polypeptides
associate in the presence of MgATP. Incubation with
[
-32P]ATP under conditions that result in
phosphorylation of the full-length Na,K-ATPase did not result in
32P incorporation into either the His6 tag or
glutathione S-transferase fusion proteins. The
MgATP-induced association was strongly inhibited by prior
modification of the fusion proteins with fluorescein isothiocyanate or
by simultaneous incubation with 10 µM eosin, indicating
that the effect of MgATP is due to interactions within the
nucleotide-binding domain. These data are consistent with Na,K-ATPase
associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for
ion transport remains unresolved, they may play a role in cell
function and in modulating interactions between the Na,K-ATPase and
other proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit that spans the plasma membrane 10 times (2) and a
~55-kDa glycosylated
-subunit that has a short cytoplasmic
N-terminal domain, a single transmembrane domain, and a large
extracellular domain. At present, whether the
-subunit plays a part
in the transport process remains unclear, but evidence is accumulating
that indicates the importance of
in targeting the enzyme complex to
the plasma membrane (3, 4). The Na,K-ATPase belongs to a large family
of enzymes known as the P2-type ATPases (5). Members of
this important protein class couple the hydrolysis of ATP to the
transmembrane translocation of cations and have been identified in
every taxonomic phylum.
-helical (8). Indeed,
the SERCA crystal structure confirmed several previously proposed
structural suggestions based on functional studies, including the
suggestion that the fifth and sixth transmembrane domains were
surrounded by the other helices as opposed to directly contacting the
membrane lipid (10, 11). In addition, the structure of the cytoplasmic
loops results in the formation of three distinct domains: 1) the N
domain, containing the nucleotide-binding site; 2) the P domain,
containing the phosphorylation site; and 3) the A domain, referred to
as the "activator" domain by Toyoshima et al. (8, 9),
consisting of the N terminus and the M2M3 loop.
and
. Consequently, these enzymes have a quaternary
structure; whether this structure is simply an
protomer or
contains higher oligomers remains a central issue of scientific
investigation (see Ref. 1). Identification of the subunit domains
involved in assembly and trafficking of Na,K-ATPase has been approached by immune precipitation experiments with truncated
-subunits (12,
13) and chimeras between the Na,K-ATPase and gastric H,K-ATPase
-subunits (14, 15).
protomers of the sodium pump are sufficient to perform Na,K-ATPase activity (16, 17). However,
it has been convincingly demonstrated that sodium pump protomer-protomer interactions do indeed take place and that adjacent pumps can be tethered together via chemical cross-linking (18). This
apparent paradox has been explained by suggesting that close packing of
sodium pumps within high density preparations leads to incidental
contact, which can be captured via cross-linking (19). Although some
functional measurements appear more easily resolved by a functional
diprotomer (20-22), more complex models for a single functioning
protomer can accommodate many of these findings (23). Nevertheless, it
is clear that sodium pump molecules are in close proximity to each
other within some cell membrane preparations. Although this association
may not be necessary for sodium pump action, it may play a role in cell
function by bringing other proteins that interact with the sodium pump
into close proximity. For example, the Na,K-ATPase has been shown to be
a membrane anchor for phosphoinositide-3 kinase in opossum kidney cells
(24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of the rat Na,K-ATPase. The M4M5 loop
includes the amino acid residues between Lys354 and
Lys774 of the
1-subunit from rat. Two fusion
protein versions of this peptide were constructed for this study: 1) a
glutathione S-transferase (GST)-tagged M4M5 loop and 2) a
His6-tagged M4M5 loop.
1
cDNA construct (generous gift from Dr. Robert Mercer, Washington
University, St. Louis, MO). An EcoRI restriction
endonuclease site was engineered into both primers, allowing a single
digestion and ligation step into the multiple cloning site of
pGEX-1
T vector (Amersham Biosciences). Positively transformed DH5
cells were selected for ampicillin resistance conferred by pGEX-1
T,
and correctly oriented clones were determined via restriction
endonuclease mapping and subsequent DNA sequencing.
-D-thiogalactoside, and the cells were grown for an
additional 12 h at room temperature. Bacterial cells were
collected by centrifugation (7500 × g for 25 min),
resuspended in 10 ml of BPER II (Pierce), and lysed via gentle
Dounce homogenization. Lysis was performed in the presence of a
protease inhibitor mixture containing 80 µM
phenylmethylsulfonyl fluoride and 10 µg/ml each of leupeptin,
antipain, and pepstatin A. After lysis, soluble proteins were separated
from cellular debris by centrifugation (13,000 × g, 45 min). The GST fusion protein was purified from the supernatant via a
glutathione-Sepharose 4B (Amersham Biosciences) affinity column, and
then the bound protein was washed with ~50 column volumes of
buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM
KH2PO4, pH 7.3) to remove nonspecifically bound
E. coli proteins. The protein was used in subsequent
procedures either bound to the support or eluted off the affinity
column with 10 mM reduced glutathione. The eluent was
dialyzed overnight against 50 mM Tris (pH 7.4) to remove
the glutathione and phosphate for interaction experiments. When
support-bound GST-protein was used, the column was washed with two
volumes of 50 mM Tris (pH 7.4) to remove the phosphate. The
size of the GST-M4M5 construct is ~63 kDa, estimated by SDS-PAGE according to the method of Laemmli (27). Protein content was determined by the method of Bradford (28). The yield was
typically 5-8 mg of GST-M4M5 loop/liter of cell culture.
1-subunit (i.e.
Lys354-Lys774) used for the GST construct was
cloned into the six-histidine fusion protein expression vector, pET-28b
(Novagen), between the EcoRI and NdeI sites. The
E. coli transformants were selected by the kanamycin
resistance (30 µg/ml) conferred by pET-28. A single colony was grown
overnight, and this culture was used to inoculate 500 ml of
LBkan (2% ethanol). After reaching an
A600 of 0.8, protein expression was induced by
the addition of a final concentration of 1 mM isopropyl
-D-thiogalactoside, and the cells were grown at room
temperature for an additional 12 h. Bacteria were collected and
lysed, and soluble proteins were separated from cellular debris via
centrifugation, as described above for the GST-M4M5 loop. The
His6-tagged ATP-binding domain was purified by passing the
lysate over a Ni2+ affinity column, His-Bind resin
(nitrilotriacetic acid-agarose, Novagen). The column was washed with
~50 column volumes of binding buffer containing 5 mM
imidazole, 500 mM NaCl, and 20 mM Tris-HCl (pH
7.9). The His6-tagged protein was eluted from the resin
with elution buffer containing 400 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9). The eluent
was dialyzed overnight against 50 mM Tris (pH 7.4) to
remove the imidazole for interaction experiments. The size of the
His6 construct is ~46 kDa, estimated by SDS-PAGE according to the method of Laemmli (27). Protein content was determined
by the method of Bradford (28).
-mercaptoethanol), and a 50-µl aliquot was resolved on a 7.5%
SDS-PAGE gel according to the method of Laemmli (27). After
electrophoresis, proteins were transferred onto PVDF membranes by
electroblotting in 10 mM CAPS, 10% MeOH, pH 11.0, for
2 h at 180-mA constant current (31). The PVDF membrane was blocked
with 10% dry milk protein solution in phosphate-buffered saline for
1 h. The membrane was then incubated with an antibody against the
Na,K-ATPase
- or
-subunit for 1 h at room temperature. The
primary antibody was removed, and the membrane was washed three times
with phosphate-buffered saline plus 0.1% Tween 20 and then incubated
for 1 h with the appropriate horseradish peroxidase-conjugated
secondary anti-IgG at room temperature. The membrane was then washed
five times with phosphate-buffered saline plus 0.1% Tween 20, and the
proteins were visualized by chemiluminescent detection of peroxidase
activity using the SuperSignal substrate kit (Pierce).
-32P]ATP
(PerkinElmer Life Sciences) and 7.3 µM Tris-ATP) and
incubated in an ice bath for 60 s. The phosphorylation was stopped
with 750 µl of "ice-cold" 5% (v/v) perchloric acid containing
0.5 mM Tris-ATP and 1.5 mM Tris-phosphate. The
samples were filtered through Millipore filters (pore size 0.45 µm),
washed three times with 3 ml of stopping buffer, and counted in a
scintillation counter. Specific phosphorylation was calculated from the
difference between 32P incorporation in native protein
preparations and those where perchloric acid was added before ATP to
denature the proteins.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
antibody and in one experiment where membrane was
probed with anti-
(Fig. 1B). In contrast, we never
observed GST-ABD/Na,K-ATPase interactions in the absence of substrates
or in the presence of sodium, potassium, or magnesium, alone.
Additionally, when experiments were performed with dog kidney enzyme
that had not been treated with C12E8 (see "Experimental Procedures"), no interactions were observed in the presence or absence of any substrates (data not shown). The observed interactions were not due to C12E8 denaturation
of the enzyme, since the detergent-solubilized enzyme has been shown to
retain function (16, 38), and we confirmed these findings for our experimental conditions (Fig. 1C).
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Fig. 1.
GST-ABD interaction with
C12E8-solubilized dog kidney Na,K-ATPase.
The GST-ABD bound to glutathione-Sepharose was incubated with 50 µg
of purified Na,K-ATPase solubilized with 0.1%
C12E8. A, SDS-PAGE. The incubation
medium contained 50 mM Tris (pH 7.4) alone
(lanes 1 and 2) or with the addition
of one of the following: 50 mM NaCl (lane
3), 50 mM KCl (lane 4), 3 mM MgAMP (lane 5), 3 mM
MgADP (lane 6), 3 mM MgATP
(lane 7), 3 mM MgPi
(lane 8). After the incubation period, the
Sepharose was washed with the corresponding media, and bound
proteins were removed by adding Laemmli sample buffer. Aliquots were
run on a 7.5% Laemmli gel, and proteins were electrotransferred to a
PVDF membrane. The membranes were probed with either anti-KETYY
antibody (upper gel) (gift from Dr. Jack Kyte,
University of California, San Diego) or anti- antibody
(lower gel) (Affinity Bioreagents). There is
clearly a strong association between the GST-ABD and the full-length
Na,K-ATPase in the presence of MgATP (lane 7) and
a somewhat weaker association in the presence of MgADP (lane
6). B, ATPase activity. There appears to be no
difference in the ability of the untreated or the
C12E8-treated Na,K-ATPase to hydrolyze ATP,
suggesting that the enzyme was not denatured.
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Fig. 2.
GST-ABD interaction with
C12E8-solubilized dog kidney Na,K-ATPase at
varying [MgATP]. The GST-ABD bound to glutathione-Sepharose was
incubated with 50 µg of purified Na,K-ATPase solubilized with 0.1%
C12E8. The incubation medium contained 50 mM Tris (pH 7.4) with the indicated concentrations of
magnesium and ATP. After the incubation period, the Sepharose was
washed with the corresponding media, and bound proteins were
removed by adding Laemmli sample buffer. Aliquots were run on a 7.5%
Laemmli gel, and proteins were electrotransferred to a PVDF membrane.
The membranes were probed with anti- antibody (Affinity
Bioreagents). The lack of protein-protein interactions at low MgATP
suggests that the GST-ABD must be in the nucleotide-bound state to
interact with the
-subunit.
-subunit.
Furthermore, it has been shown that this domain undergoes dramatic
conformational changes in response to substrate binding (39, 40).
Consequently, we decided to test whether the interactions observed
between the intact Na,K-ATPase and the GST-ABD were mediated via
contacts within the ATP-binding domain. These experiments were
performed using either the GST-ABD or a His6-tagged fusion
protein with the same polypeptide as the GST-ABD. The
nucleotide-binding properties of the His6-ABD were
described previously (26); in addition, we found that the
His6-ABD, like the GST-ABD, associates with the intact
Na,K-ATPase in a MgATP-dependent manner (Fig.
3). Therefore, the observed interactions
between native Na,K-ATPase and the fusion proteins were mediated by
enzyme contacts with the ATP-binding domain and not by either of
the fusion protein affinity tags.
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Fig. 3.
His6-ABD interaction with
C12E8-solubilized dog kidney Na,K-ATPase.
The His6-ABD bound to Ni2+-nitrilotriacetic
acid was incubated with 50 µg of purified Na,K-ATPase solubilized
with 0.1% C12E8. The incubation medium
contained 50 mM Tris (pH 7.4) with or without 3 mM MgATP. After the incubation period, the
Ni2+-nitrilotriacetic acid was washed with the
corresponding media, and bound proteins were removed by adding
Laemmli sample buffer. Aliquots were run on a 7.5% Laemmli gel, and
proteins were electrotransferred to a PVDF membrane. The membranes were
probed with anti-KETYY antibody. It is clear that the presence of MgATP
dramatically increased the amount of Na,K-ATPase bound to the
His6-ABD.
-subunit elicits a
conformation slightly different than when it is bound to the isolated
M4M5 loop alone. Indeed, the nucleotide-bound crystal structure of
SERCA indicates that both the N terminus and the M2M3 cytoplasmic loop
(i.e. the "A" domain) are in close proximity to the M4M5
loop (8, 9); the A domain is obviously absent from our bacterial
constructs.
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Fig. 4.
Binding between the GST-ABD and
His6-ABD is elicited by MgATP. GST-ABD tethered to
glutathione-Sepharose was incubated with a 50-µg quantity of
His6-ABD as described under "Experimental Procedures."
A, separately, incubations contained 5 mM ATP,
AMP, Pi, or TBS in the absence of Mg2+ ions
(left) or combined with 5 mM Mg2+
(right). B, a separate experiment is shown that
also included 5 mM MgADP. Both blots were probed with
anti-penta-His antibody (Qiagen). Clearly, both magnesium and ATP
together produce the most stable interaction between the two
constructs.
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Fig. 5.
Specificity of the GST-ABD for binding the
His-ABD. In order to determine whether there were significant
nonspecific interactions between bacterially purified fusion proteins
and the GST-ABD and/or the glutathione resin, we used an unrelated
protein, streptavidin-tagged SOD, in identical interaction experiments.
Clearly, there was no interaction between SOD and the GST-ABD in the
presence or absence of ATP. As a positive control for immunodetection,
we ran 1 µg of purified SOD in an adjacent lane
(left).
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Fig. 6.
Inhibition of MgATP binding by competition
with eosin. GST-ABD tethered to glutathione-Sepharose was
incubated with a 50-µg quantity of His6-ABD in the
presence of magnesium and ATP in the presence and absence of 10 µM eosin. Lane 1, interactions
between the ABDs in the presence of 1 mM MgATP and 10 µM eosin. Lane 2, interactions
between the ABDs in the presence of 1 mM MgATP alone. Both
the Coomassie-stained gel (left panel) and the
immunoblot (right panel) indicate much smaller
amounts of the His6-tagged loop associated with the GST
fusion protein in the presence of eosin. His6-ABD protein
was detected with anti-penta-His antibody (Qiagen). Experiments were
performed essentially as in Fig. 5 with the addition of eosin.
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Fig. 7.
Effect of FITC modification on MgATP-induced
ABD associations. These experiments were designed to determine
whether FITC modification of both the GST- and
His6-ABDs was required to prevent the
MgATP-dependent association. For FITC modification, the
fusion proteins were treated with 20 µM FITC for 30 min
at room temperature (50 mM Tris, pH 9.0). Unreacted FITC
was removed via dialysis (see "Experimental Procedures").
A, Unlabeled GST-ABD, tethered to glutathione-Sepharose, was
incubated with a 50-µg quantity of either unlabeled
His6-ABD or FITC-labeled His6-ABD. Interactions
were measured in the presence of 1 mM MgATP. Equal aliquots
from the respective interactions were run in separate lanes on a 12%
Laemmli gel. The gel was cut in half, and one section was used for
Western analysis with anti-penta-HIS antibody (right
panel), whereas the other half was first photographed under
UV illumination (middle panel) and then stained
with Coomassie Brilliant Blue (left panel).
Lane 1 in each panel shows
interactions in the complete absence of FITC labeling. Lane
2 in each panel shows the interaction between
unlabeled GST-ABD and FITC-labeled His6-ABD. Clearly,
FITC-modified His6-ABD was still able to associate with
unlabeled GST-ABD in the presence of 1 mM MgATP
(right panel, lane 2).
B, FITC-labeled or unlabeled GST-ABD, tethered to
glutathione-Sepharose, was incubated with a 50-µg quantity of either
unlabeled His6-ABD or FITC-labeled His6-ABD.
Interactions were measured in the presence of 1 mM MgATP.
Equal aliquots from the respective interactions were run in separate
lanes on a 12% Laemmli gel. The gel was cut in half, and one section
was used for Western analysis with anti-penta-His antibody
(right panel), whereas the other half was first
photographed under UV illumination (middle panel)
and then stained with Coomassie Brilliant Blue (left
panel). Lane 1 in each panel shows
interactions when both fusion proteins were labeled with FITC.
Lane 2 is the control, showing normal association
in the complete absence of FITC modification. Clearly, FITC
modification of the fusion proteins dramatically reduced their ability
to associate with one another (right panel,
lane 1).
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Fig. 8.
Phosphorylation of the Na,K-ATPase and
bacterially expressed domains by [32P]ATP. Purified
His6-ABD, dog kidney Na,K-ATPase, and GST-ABD were
incubated with [32P]ATP on ice as described under
"Experimental Procedures." 32Pi associated
with protein denatured a priori with perchloric acid was
subtracted from nondenatured protein, and the difference is shown in
the figure as specific 32Pi
incorporation. There appears to be no covalent transfer of phosphate to
either fusion protein, whereas native Na,K-ATPase was readily
phosphorylated. Data represent means, and bars represent the
S.E. from three experiments with triplicate determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit (Fig.
2). These observations are consistent with the necessity of ATP binding
to both interacting proteins to facilitate association, or
alternatively, they may reflect a necessary
MgATP-dependent stabilization or decrease in flexibility of
the soluble GST-ABD to achieve this stable protein-protein interaction.
-subunit. Since
FITC binding to both partners is required to prevent association,
either partner can supply this segment. The other partner may provide
the terminal phosphate binding segment (close to Asp369) in
the P domain. In this way, the ATP molecule bridges two ABD polypeptide
loops via N and P domains from each member of the dimer (see below).
/
multimer (i.e.
(
)n). Initially, (
)2 models were
proposed to explain the biphasic kinetics of ATP on enzyme activity
(47) and later expanded to (
)4 models (48).
Structural support for oligomeric models comes from a number of diverse
studies, including cross-linking (18), fluorescence resonance energy
transfer between the ATP-site probes FITC and ErITC (22), and more
directly by co-immunoprecipitation experiments (49).
-
dimer and tetramer aggregates (50). Interestingly, these oligomers were devoid of
- and
-subunits, demonstrating that the important contacts were within the
-subunit (50).
-subunit isoforms (i.e.
1 and
3) heterologously expressed in insect cells, via
co-immunoprecipitation experiments with isoform-specific antibodies. In
these experiments, the expressed Na,K-ATPase is less than 10% of the
total membrane protein and most likely closer to 1-2% (3). Clearly,
the pump density in insect cell preparations is sufficiently low that
-
interactions are unlikely to have been caused by overcrowding.
In the present study, the ability of both GST- and His6-ABD
to pull down intact
-subunit from a C12E8-solubilized kidney preparation of
Na,K-ATPase (Figs. 1 and 3) in the presence of defined ligands is more
consistent with specific interactions than incidental contact. Indeed,
the lack of interaction between the ABDs and intact Na,K-ATPase
-subunit, in the absence of solubilization, suggests that endogenous
pump-pump associations must be broken before the exogenous ABDs can
bind. Moreover, the demonstration that the two tagged ABDs associate with the same substrate dependence as with the intact Na,K-ATPase indicates that pump-pump interactions may be solely through
interactions between the large cytoplasmic loops. Indeed, Koster
et al. (45) reached similar conclusions based upon
-
associations measured between different Na,K- and H,K-ATPase chimeric
enzymes expressed in insect cells. Specifically, the sodium pump
1 subunit selectively associated with chimeras
containing the Na,K-M4M5 loop but not with constructs containing the
H,K-ATPase large cytoplasmic loop (45). In fact, these authors observed
that wild-type sodium pump
1 associated with a chimera
containing only the sodium pump M4M5 loop inserted within the
H,K-ATPase (45).
-
Interactions--
It seems clear that the
catalytic properties of Na,K-ATPase can be mediated by a functional
monomeric form (i.e.
) of the enzyme (16, 19, 23, 38).
Consequently, the relevance of the higher oligomeric states sometimes
proposed for this enzyme remains a puzzle. There are several reports
measuring various kinetic properties of Na,K-ATPase function that can
be easily explained by
-
interactions. For example, fluorescence
resonance energy transfer between several Na,K-ATPase labeling probes
reveals distances great enough to indicate that the probes reside on
different protomers (22). These observations are directly in contrast with FRET measurements from another laboratory indicating that FITC and
Co(NH3)4ATP are close enough to reside on a
single
protomer (52). In addition, the ability of TNP-ADP to
inhibit the residual p-nitrophenyl phosphatase
activity of FITC-labeled
protomers, solubilized with
C12E8, makes it difficult to justify invoking
the involvement of additional protomers (21). Indeed, Martin and Sachs
(23) convincingly demonstrate that the appearance of the "low
affinity" nucleotide site, which allows TNP-ADP to bind and inhibit
p-nitrophenyl phosphatase activity, is actually a
result of FITC modification and does not appear on native enzyme. Thus,
there seems to be no compelling functional reason to suggest that the
Na,K-ATPase exists as a diprotomer (or higher oligomer). Nevertheless,
the current observations (Figs. 1-3) as well as previous reports from
other laboratories (18, 45, 48-50) strongly suggest that under certain
conditions the Na,K-ATPase does self-associate.
-subunit. However, an intermolecular interaction might
account for our observed GST-ABD/His6-ABD association. Our observations that FITC modification was required of both fusion proteins to disrupt the interaction suggest that eliminating MgATP binding to one of the partners alone was without effect. In the native
protein, it seems likely that following MgATP binding, closure of P and
N domains occurs, facilitated by changes in the intramembrane helices
and the A domain. Since the GST-ABD and His6-ABD are devoid
of both the transmembrane segments and A domains, they would be unable
to close upon themselves (i.e. an intramolecular N and P
interaction cannot occur within either fusion protein). Rather, MgATP
binding induces a conformational change in the N domain within one
fusion protein, but the P domain for the interaction is supplied from
another molecule that interacts. Thus, FITC modification of one partner
would not eliminate interactions, since that partner would supply the P
domain for the interaction (see above).
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully thank Phillip Weeks for excellent technical assistance. We thank Drs. Dwight Martin and John Sachs (SUNY-Stoneybrook) for fruitful discussions of this work and Dr. Michael Caplan (Yale University) for sharing unpublished findings. In addition, we thank Dr. Mark Milanick (University of Missouri) for helpful comments and for the use of his laboratory for the phosphorylation experiments. We also thank John Eisses for providing the streptavidin-tagged SOD construct for our control experiments (Fig. 5).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grant GM61583 and American Heart Association Grant 0030161N (to C. G.) and NIH Grant GM39500 (to J. H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, L224, Oregon Health & Science
University, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-1001; Fax: 503-494-1002; E-mail: kaplanj@ohsu.edu.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M212351200
2 M. Caplan, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SERCA, sarcoplasmic reticulum Ca-ATPase; ABD, ATP-binding domain; FITC, fluorescein 5'-isothiocyanate; PVDF, polyvinylidene difluoride; SOD, superoxide dismutase; TBS, Tris-buffered saline; GST, glutathione S-transferase; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
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