Program in Biophysics, Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester New York 14642
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ABSTRACT |
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Although its primary function is monovalent anion exchange, the
band 3 protein also cotransports divalent anions together with protons
at low pH. The putative proton binding site, Glu-681 in human
erythrocyte band 3, is conserved throughout the anion exchanger family
(AE family). To determine whether or not the monovalent anion binding
site is located near Glu-681, we modified this residue with Woodward's
reagent K
(N-ethyl-5-phenylisoxazolium-3'-sulfonate; WRK). Measurements of
Cl binding by
35Cl-NMR show that external
Cl
binds to band 3 even
when Cl
transport is
inhibited ~95% by WRK modification of Glu-681. This indicates that
the external Cl
binding
site is not located near Glu-681 and thus presumably is distant from
the proton binding site. DIDS inhibits
Cl
binding even when WRK is
bound to Glu-681, indicating that the DIDS binding site is also distant
from Glu-681. Our data suggest that the DIDS site and probably also the
externally facing Cl
transport site are located nearer to the external surface of the
membrane than Glu-681.
anion exchangers; anion transport; red blood cells; erythrocytes; nuclear magnetic resonance; anion exchanger 1
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INTRODUCTION |
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BAND 3, OR AE1, a 101.7-kDa
protein found in the erythrocyte membrane, is a member of the AE family
of anion exchangers. Under physiological conditions band 3 catalyzes
the one-for-one, electroneutral exchange of
Cl and bicarbonate. The
protein also mediates the exchange of divalent anions such as sulfate,
a process that is accelerated at low extracellular pH (4, 10); the
transport of sulfate has been shown to be accompanied by the
cotransport of a proton (15).
The zwitterionic reagent Woodward's reagent K
(N-ethyl-5-phenylisoxazolium-3'-sulfonate;
WRK) reacts with carboxyl groups; the typical targets of its effects in
proteins are aspartate and glutamate. Jennings and Anderson (18) have
shown that when intact red blood cells are treated with WRK at
22°C, there is no detectable reaction with aspartate residues in
band 3, as detected by labeling the reaction products with
NaB3H4.
At this temperature, both the
NH2-terminal 60-kDa and the COOH-terminal 35-kDa chymotryptic fragments of band 3 are labeled by
WRK plus BH4, but when the
reaction is run near 0°C, almost all the labeling is in the 35-kDa
fragment. The label in this fragment is almost exclusively on Glu-681
(19). This result is surprising, because another glutamate, Glu-658, is
probably located much nearer to the external membrane surface than
Glu-681 (34). Conversion of the Glu-681 carboxyl group to an alcohol by
WRK plus BH4 inhibits
Cl/Cl
exchange and accelerates sulfate/sulfate exchange, sulfate influx into
Cl
-containing cells (17),
and sulfate efflux into
Cl
-containing media (16).
This modification of Glu-681 affects both the intracellular and
extracellular pH dependence of band 3-mediated sulfate flux, suggesting
that Glu-681 can cross the anion permeation barrier in band 3 and that
it is the residue that binds protons in proton-sulfate cotransport
(17).
Cotransport of protons and sulfate across the permeability barrier is consistent with a model in which the proton and sulfate binding sites are located near each other in a substrate "pocket" within the protein. The observation that external proton binding increases external sulfate binding affinity and vice versa (26) supports such a picture, although a model in which the binding sites are allosterically coupled is also consistent with this evidence. The increase in sulfate transport observed when the carboxyl group on Glu-681 is converted to the corresponding alcohol by treatment with WRK and BH4 (17) supports the hypothesis that the sulfate binding site is located near Glu-681. Consistent with this idea, modification by WRK and BH4, like external proton binding, also increases external sulfate affinity (M. L. Jennings, personal communication). Mutational studies by Sekler et al. (32) and Chernova et al. (3) with mouse band 3 also implicate Glu-699, the murine analog of Glu-681, in the control of sulfate transport.
Sulfate and Cl are mutually
competitive inhibitors of each other's transport (8, 27, 31), and
sulfate exchange and Cl
exchange are inhibited to the same extent by various concentrations of
different transport inhibitors (21). This has led to the idea that the
monovalent anion binding site is located in a pocket within the band 3 structure near, or perhaps coincident with, the proton and divalent
anion binding sites (26). However, other experiments have shown that
the inhibitor
N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate interacts differently with
Cl
than with
SO2
4 (8), suggesting that monovalent and divalent substrates may not necessarily bind to the same site in
band 3.
Kinetic experiments with I,
a slowly transported monovalent anion substrate, also support a model
in which monovalent and divalent anions bind to different sites. Most
of the kinetic data for band 3 can be fitted to a lock-carrier
ping-pong model. According to this model, there are two different
conformations of the protein: Eo,
in which the transport site is accessible to external
Cl
, and
Ei, in which
Cl
can bind from the inside
or from the cytoplasm. Once
Cl
is bound, the protein
can undergo the "transporting" conformational change from
Eo to
Ei or vice versa. Milanick and
Gunn (27) determined the dissociation constants
(Kd) for the
binding of an external proton to the outwardly facing transport site,
as well as to this transport site when iodide or sulfate is bound. They
found that the Kd
for external H+ binding to the
protein in Eo was 1 × 10
5 M. When sulfate is
bound to the protein in Eo, the
external-proton Kd is reduced to
1 × 10
6 M, consistent
with a picture in which sulfate and
H+ are cotransported and the
binding of one ion increases the binding affinity of the other (26).
The surprising result, however, is that when
I
is bound to
Eo the external proton
Kd increases to
1.2 × 10
5 M.
Milanick and Gunn point out that this increase in the proton
dissociation constant is contrary to what would be expected from simple
electrostatic arguments if the monovalent anion and sulfate/proton binding pockets overlapped. If
I were bound in the same
position as sulfate, the electrostatic interaction predicts that the
Kd for external
H+ with
I
bound should decrease to
3.2 × 10
6
M. Further supporting the conclusion that the binding sites for monovalent and divalent anions are not identical, the same authors have
observed that internal protons can inhibit the translocation of
Cl
by binding to band 3 when the Cl
transport site
is in the unloaded, externally facing form
(Eo). This implies that an
internal proton can bind band 3 in its
Eo conformation. However, the
complex of an internal Cl
bound to band 3 in Eo is never
observed, which suggests that different gating mechanisms regulate
proton and monovalent anion binding and that the two ions' binding
sites are distinct (27). This conclusion, however, is based on the
assumption that the proton that inhibits
Cl
transport binds to the
same site as the proton involved in
H+-SO2
4
cotransport. In other words, this interpretation assumes a single
H+ binding site. We address the
implications of this assumption in more detail below.
In light of these conflicting observations, it is of great interest to
obtain structural as well as kinetic information about the proximity of
Cl and
H+ binding sites in band 3. However, structural information from two-dimensional crystals of band 3 (35, 36) so far has insufficient resolution to identify putative
substrate binding sites. In the present paper, we examine
the relation of the Glu-681 residue to the monovalent substrate binding
site by studying the effects of a structural modification of band 3, the reaction of WRK with Glu-681, on
Cl
binding by
35Cl-NMR.
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THEORY |
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Measurement of Cl binding to the
external transport site of band 3 by
35Cl-NMR.
The
35Cl
nucleus has spin 3/2, and thus there are four nuclear spin states, 3/2,
1/2,
1/2, and
3/2. The binding of
35Cl
to macromolecules such as band 3 can be observed by NMR because the
Cl
line width is much
larger when the anion is bound than when it is free in solution. The
observed
35Cl
spectra are due to a broadening of the spectral line width of free
Cl
in solution due to
exchange of free Cl
with
Cl
in the bound state (2).
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(1) |
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(2) |
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(3) |
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(4) |
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(5) |
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MATERIALS AND METHODS |
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Cell preparation. Fresh blood samples were obtained from apparently healthy human donors, with Na+-heparin as the anticoagulant. Cells were washed twice in an ice-cold solution of PBS (150 mM NaCl, 20 mM NaH2PO4, pH 6.0) to remove plasma, white blood cells, and fibrin and then twice in 150 KH medium (in mM: 150 KCl, 20 HEPES, 28 sucrose, 5 glucose; pH 7.04 at room temperature). Cells were brought to a concentration of 50% hematocrit (Hct) in 150 KH medium.
Measurement of
36Cl
efflux.
Cells were loaded with
36Cl
by incubation at 25% Hct in 150 KH medium, with a trace amount of
36Cl
added (usually to a final concentration of 2.5 µCi/ml for 25% Hct
cells). Cells were incubated for 10 min at room temperature and then
placed on ice.
WRK treatment. Red blood cells were treated with WRK in a manner similar to that of Jennings and Al-Rhaiyel (17). Cells were prepared as described above, but after washes with PBS and 150 KH medium the cells were resuspended to 10% Hct in ice-cold 150 KH medium. Solid WRK was added to the cell suspension to give a final concentration of 2 mM (calculated on the basis of the total volume of the cell suspension). The cell suspension was gently but well mixed and incubated on ice for 10 min. Cells were then spun down, washed, and resuspended for loading with the radioisotope.
35Cl-NMR. Cells were prepared in the same manner as for radioisotope flux experiments, and 3.5- to 4.0-ml samples of cells at 50% Hct were placed in 7-in. glass NMR sample tubes (Wilmad Glass, Buena, NJ). Cells were treated with WRK as described above. For the DIDS-treated NMR samples, DIDS (Molecular Probes, Eugene, OR) was added to a concentration of 100 µM (in the total volume of cell suspension) and cells were incubated at 25% Hct at room temperature (and shielded from light) for 30 min. They were then washed once with 150 KH medium and resuspended to 50% Hct before being placed in the NMR sample tube. The length of time between the addition of cells to the sample tube and NMR measurement of the sample was kept to a minimum to avoid lysis, which can cause artifactual increases in LBf. We also attempted to decrease lysis by minimizing the amount of time the cells were kept at high Hct.
All NMR measurements were performed on a 9.4 T Bruker/GE Omega NMR spectrometer (Bruker Instruments, Fremont, CA). The spectrometer operated at a frequency of 39.2 MHz for 35Cl and 400 MHz for protons. A 10-mm-diameter broad-band tunable probe was used, and the sample was kept at a constant temperature by a flow of nitrogen gas regulated at 3°C over the sample. The sweep width was 2,000 Hz for buffer samples (i.e., samples not containing erythrocytes) and 4,000 Hz for cell samples; 512 points were collected per scan. Shimming was done on the water proton signal for each sample; the solvent proton line width was ~3-5 Hz for buffer and 11 Hz for red blood cell samples. Typically, 5,000 data acquisitions were averaged, requiring a total time of 740 s. As described in THEORY, the magnitude of the FID signal was fitted to a biexponential function (Eq. 1) by the method of least squares with software provided with the instrument.DQF NMR.
35Cl
DQF signals were generated with the following pulse sequence (1, 10):
0°-
/2-180°-
/2-90°-
-90°-Acq where
is the creation time and
is a 2-µs delay to reset the radio
frequency phase. The pulse and receiver phase cycling scheme was that
given by Bax et al. (1).
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RESULTS |
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Effect of WRK treatment on Cl
transport.
Typical effects of WRK treatment on
Cl
/Cl
exchange are presented in Fig.
1, which shows the rate
constants, k, for
Cl
exchange under various
conditions divided by the rate constant for control cells,
kc. Cells were
treated with WRK on ice as described in MATERIALS AND
METHODS and then loaded with
36Cl
.
Efflux took place into 150 KH medium or 150 KH medium with 10 µM
DIDS. The reaction with WRK inhibited
Cl
efflux by 96 ± 4%
(mean ± SD), a larger percentage of inhibition than
that reported by Jennings (16), who observed 79% inhibition. The
higher inhibition may be related to the slightly higher pH in our
experiments.
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Effect of WRK on external Cl
binding.
To approach the question of the proximity of the external
Cl
binding site to Glu-681,
we measured Cl
binding to
band 3 by 35Cl-NMR in erythrocytes
treated with WRK. If the reaction with WRK prevents
Cl
binding, this would
indicate that the Cl
binding site lies in close proximity to Glu-681 or is affected allosterically by the reaction of WRK with Glu-681. If, however, Cl
binding still takes
place in cells that have been treated with WRK, this would mean that
the Cl
binding site is not
located near Glu-681. Note that this method does not provide us with a
means of measuring the precise distance between the
Cl
and WRK, so the
conclusion that Cl
is or is
not located near Glu-681 must be taken as a qualitative statement. The
degree to which any quantitative conclusions can be drawn about the
separation between the Cl
binding site and Glu-681 is considered in discussion.
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Effect of WRK on Cl affinity.
The decrease in DIDS-sensitive LBf
in WRK-treated cells might reflect a decrease in affinity for external
Cl
(see
Eq. 4). In Fig.
3 we show
35Cl
LBf measured in WRK-modified cells
in media containing different values of external
Cl
concentration
([Cl
]o)
at 0°C. A fit to these data gives an apparent
Kd value of 44 ± 16 mM for Cl
binding
to the external site in WRK-modified band 3. The implications of this
value in terms of effects of WRK treatment on
Cl
affinity are considered
in discussion. It is immediately
apparent from Eq. 4, however, that,
even if the Kd
value for control cells were negligibly small, the measured
Kd for
WRK-treated cells would predict only a 23% decrease in
LBf for 150 mM
[Cl
]o;
thus changes in
Kd are not
entirely responsible for the decrease in DIDS-sensitive
LBf seen in Fig. 2.
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DQF experiments.
We have previously shown that the external
Cl transport site of band 3 is the only external site that substantially restricts the motions of
Cl
and that is present in
sufficient quantity to give rise to an observable DQF signal (25).
Although the DQF signal is not linearly related to the extent of
Cl
binding, as is the
LBf, it provides a useful
qualitative test for band 3-related
Cl
binding that is
independent of possible problems associated with the double-exponential
fitting process that is required to analyze the FID data shown in Fig.
2. Figure 4 shows the DQF signals from control (A), DIDS-treated
(B), and WRK-treated
(C) cells, as well as from cells
treated with both WRK and DIDS (D).
Note that the DQF signal is present in control (untreated) cells, but
not in the 150 mM Cl
buffer
(E) or in DIDS-treated
(B) red blood cells. The
absence of a DQF signal in DIDS-treated cells demonstrates that the
signal arises from a DIDS-protected site(s), presumably the external anion transport site(s).
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DISCUSSION |
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The studies described here reveal a clearer picture of the relation
between various binding sites on the band 3 protein. As we have
discussed, much previous evidence has been consistent with a simple
model in which all band 3 substrates, protons, divalent anions, and
monovalent anions, bind to nearby sites in a pocket of the protein
possibly close to Glu-681. Our studies, however, support the suggestion
of Milanick and Gunn (27) that the relationship between monovalent
anion binding sites and the proton binding site is not so simple. Our
NMR results are not consistent with a model in which
Cl binds near the site of
the WRK covalent reaction, and thus presumably are inconsistent with a
model in which Cl
binds
near Glu-681, the putative proton binding site.
Specificity of WRK for Glu-681.
Jennings and Smith (19) have shown that Glu-681 is the major site of
reaction of WRK if the reaction is carried out on ice near neutral pH,
but they have also presented evidence that WRK can react with other
sites, particularly if the reaction takes place at higher temperature
(18) or if red cells are exposed to WRK multiple times (16). Because
the present data show the lack of an effect of WRK on
Cl and DIDS binding, a
reaction at other sites should not affect the conclusion that the WRK
reaction at Glu-681 does not affect these processes, so long as there
is good evidence that Glu-681 has actually been modified by WRK.
Indeed, on the basis of tritiated borohydride cleavage of the WRK
reaction products, Jennings and Smith (19) have shown that the reaction
on ice labels almost exclusively the 35-kDa COOH-terminal segment of
band 3 and that Glu-681 is selectively labeled compared with other
glutamate residues in this segment. However, the reaction at other
sites could affect the interpretation of some of the results presented
here. For example, if WRK can react with other sites on some band 3 molecules and if this reaction interferes with DIDS binding but does
not prevent Cl
binding to
the external transport site, this would explain why a DIDS-insensitive
component of the DQF NMR signal (Fig.
4D) is seen after WRK treatment.
Distance between Glu-681 and the
Cl binding site.
The reaction of Glu-681 with WRK results initially in the formation of
an active ester, which then spontaneously converts to an
N-acyl derivative (17). Because of
conformational flexibility in these adducts, neither completely
prevents access to the original glutamate side chain, but either would
occupy most of the space within ~5-6 Å of the carboxyl
carbonyl group (Fig. 5). Because one end of
the adduct formed contains a sulfonic acid group and because the adduct
is likely to be formed within a fairly narrow polar "vestibule"
within the band 3 protein, it seems unlikely that the
Cl
binding site could be
closer than ~6 Å to Glu-681, and the distance is likely to
be considerably longer because of the expected electrostatic repulsion
between Cl
and the
sulfonate moiety of WRK. It is not possible, however, to specify the
separation distance precisely from our data, and it should be
emphasized that the diagram shown in Fig. 5 is only a schematic
representation and is not intended as a precise model for the exact
location of WRK within the band 3 vestibule.
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Relation to the work of Milanick and Gunn.
The conclusions of Milanick and Gunn (27) depended in part on the
assumption that there is only a single proton binding site and that
this site is responsible for both
H+-SO24
cotransport and proton inhibition of monovalent anion transport.
Working with site-directed mutants of mouse band 3 expressed in
Xenopus oocytes, however,
Müller-Berger et al. (28) found that mutations at Glu-699 (the
murine equivalent of Glu-681) and His-752 (equivalent to His-734 in
human band 3) have almost identical effects on the external pH
dependence of Cl
exchange.
This raises the possibility that there may be more than one
protonatable site in band 3 that affects the pH dependence of anion
exchange, thereby shedding doubt on Milanick and Gunn's single-site
assumption and the conclusions drawn from it. However, it is possible
that His-752 and Glu-699 could act in a cooperative manner to provide a
site for proton binding. In such a scenario, the mutation of either
residue might disrupt normal proton binding at a single site,
consistent with the postulate of Milanick and Gunn.
Effects of WRK on Cl transport and
Cl
binding.
Our results indicate that the covalent binding of WRK to Glu-681 does
not prevent external Cl
binding, although it does prevent
Cl
transport. The
inhibition of anion transport under conditions in which substrate
binding is not prevented implies that WRK prevents the translocation
event, and thus it must interfere with at least one essential element
of the change in band 3 conformation (lock-carrier gating) that causes
the change in orientation of the
Cl
transport site from
inside to outside or vice versa. Even if Glu-681 is located at a
significant distance from the
Cl
binding site, the change
in band 3 conformation caused by the WRK reaction must affect the
gating regions that are likely to be near the transport site, and thus
may have a subtle allosteric effect on the environment of
Cl
bound to the externally
facing transport site.
Relationship of Glu-681 to the DS binding sites.
We find that the reaction with WRK does not prevent DIDS from
inhibiting Cl binding, and
hence the DIDS binding site, as well as the
Cl
binding site, must be
some distance from Glu-681. This suggests that Glu-681 is far enough
from both the Cl
binding
site and the DIDS binding site to allow DIDS to inhibit Cl
binding even after
formation of the WRK-Glu-681 adduct. This conclusion is supported by
Jennings and Anderson's observation that reductive methylation affects
3H2-DIDS
labeling but not
WRK-B3H4
labeling (18) and by Jennings's evidence (M. L. Jennings, personal
communication) that reaction of Glu-681 with WRK does not prevent
covalent
3H2-DIDS
labeling. On the other hand, Jennings and Anderson (18) have shown that WRK modification of band 3 (measured by
B3H4-WRK
labeling of band 3 or by WRK inhibition of monovalent anion exchange)
is greatly inhibited when red blood cells have been treated first with
DS such as DNDS or H2-DIDS. This
is consistent with a model in which the DS binding site is located
closer to the external face of the membrane than Glu-681. Thus DS
binding prevents external WRK from reaching Glu-681, but the reaction of WRK with Glu-681 does not prevent DS from gaining access to their
binding site.
Location of the Cl binding site
and the WRK site relative to the inner surface of the membrane.
The concept that Glu-681 is located nearer to the cytoplasmic surface
of the band 3 protein than the DIDS site is consistent with recent
cysteine-scanning mutagenesis experiments of Tang et al. (34) with
human band 3 expressed in HEK-293 cells. These experiments show that
Glu-681 is located in a region of band 3 that is protected from access
to polar reagents, such as biotin maleimide, and that presumably
corresponds to a transmembrane
-helix. Toward the
NH2 terminus, the nearest residue
in this segment that is accessible to biotin maleimide is Met-663,
which must be at the external side of the apolar part of the membrane, because the entire region between it and the external
N-glycosylation site (Asn-642) is
accessible to polar reagents. Toward the COOH-terminal side of Glu-681,
the first accessible residue is Ile-684, indicating that Glu-681 is
only three amino acids from the interior side of the apolar portion of
the membrane.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Barry Goldstein's assistance in preparing Fig. 5.
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FOOTNOTES |
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Financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-27495 and Shared Instrumentation Grant RR-06252.
C.T. Gunter was the recipient of a Strong Children's Research Summer Fellowship.
Portions of this paper have appeared previously in abstract form (20).
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. §1734 solely to indicate this fact.
1 Equation 5 predicts a nonlinear relationship between the DQF signal and LBf. In particular, at high LBf values, such as in control cells, decreases in LBf correspond to smaller fractional changes in the DQF signal. For example, for three measurements under the conditions of the DQF experiments in Fig. 4, the LBf for WRK-treated cells was an average of 59% of the control LBf. The WRK-treated cell/control cell DQF signal ratio predicted from Eq. 5, based on the LBf and LBs (line broadening of T2s) measured from the FID in the same cell samples, was 75 ± 4% (mean ± SD), which was not significantly different (P = 0.94) from the measured value of 74 ± 13%.
Address for reprint requests and other correspondence: S. Bahar, Dept. of Physics, Box 90305, Duke Univ., Durham, NC 27708 (E-mail: bahar{at}phy.duke.edu).
Received 1 February 1999; accepted in final form 16 June 1999.
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