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Contribution of the Band 3-Ankyrin Interaction to Erythrocyte Membrane
Mechanical Stability
By Philip S. Low, Barry M. Willardson, Narla Mohandas, Mary Rossi, and Stephen Shohet
In an effort to evaluate the role of the band 3-ankyrin linkage
in maintenance of red blood cell membrane integrity, solution conditions were sought that would selectively dissociate the band 3-ankyrin linkage, leaving other membrane
skeletal interactions intact. For this purpose erythrocytes
were equilibrated overnight in nutrient-containingbuffers at
a range of elevated pHs and then examined for changes in
mechanical stability and membrane skeletal composition.
Band 3 was found to be released from interaction with the
membrane skeleton over a pH range (8.4 to 9.5) that was
observed to dissociate the band 3-ankyrin interaction in
vitro. In contrast, all other membrane skeletal associations
appeared to remain intact up to pH 9.3, after which they were
S
INCE THE discovery of ankyrin’-6and its high affinity
for the membrane-spanning protein band 3,’38it has
been generally accepted that the predominant linkage
connecting the membrane skeleton to the lipid bilayer is
maintained by an interaction between these two proteins?”
However, despite compelling support for this argument,
little direct evidence exists to demonstrate the essential
contribution of the band 3-ankyrin association to overall
red blood cell (RBC) membrane stability. In the past,
clinical studies of patients with mutations in membrane
skeletal components have been used to define the importance of specific membrane interactions. However, in the
case of the band 3-ankyrin association, relatively few
aberrant linkages have been identified, probably because a
major defect in the interaction may be lethal. One relevant
study has shown that patients with a particular hemolytic
poikilocytic anemia exhibited a reduction in the number of
high-affinity membrane binding sites for ankyrin.” Another
investigation has shown an inability of mouse erythrocytes
partially deficient in ankyrin to undergo normal echinocytic
or stomatocytic shape transformation^.'^ However, cells
with highly defective ankyrin-band 3 linkages have still not
been found in nature.
To more directly examine the importance of the band
3-ankyrin attachment to overall erythrocyte stability, we
have explored the effect of artificially dissociating the
interaction on membrane fragility. While recent studies
have shown that dissociation of the two components can be
promoted in vitro by certain sulfhydryl reagents14 and by
competing antib~dies,’~
the most accessible method to
disjoin ankyrin from band 3 was found to be achieved by
simply equilibrating the pH to near 9.16 Thus, it was
demonstrated that the ankyrin-band 3 interaction is controlled in vitro by structural transitions in band 3 at pH 7.2
and 9.2, and that the latter transition reversibly converts the
anion transporter from a binding conformation to a nonbinding conformation. In this report we show that elevation of
intracellular pH above 8.5 gradually releases band 3 from
the membrane skeleton, leaving the remaining skeletal
interactions intact, and that this disconnection of the
membrane skeleton from the lipid bilayer renders the
erythrocyte membrane mechanically unstable.
Blood, Vol77, No 7 (April I), 1991:pp 1581-1586
also seen to dissociate. Whereas hemolysis of mechanically
unstressed cells did not begin until -pH 9.3, where the
membrane skeletons began to disintegrate, enhanced fragmentation of shear stressed membranes was seen to begin
near pH 8, where band 3 dissociation was first observed.
Furthermore,the shear-inducedfragmentationrate was found
to reach a maximum at pH 9.4, ie, where band 3 dissociation
was essentially complete. Based on these correlations, we
hypothesize that the band 3-ankyrin linkage of the membrane skeleton to the lipid bilayer is essential for red blood
cell stability in the face of mechanical distortion but not for
cellular integrity in the absence of mechanical stress.
o 1991 by The American Society of Hematology.
EXPERIMENTAL PROCEDURES
Materials
Triton X-100 was purchased from Boehringer Mannheim (Indianapolis, IN) and diisopropylflourophosphate was from Sigma (St
Louis, MO). Fresh blood was obtained from the Central Indiana
Regional Blood Bank or was drawn from healthy donors into acid
citrate dextose. Antibodies were raised in rabbits against purified
human erythrocyte membrane proteins and characterized as described previo~sly.’~
Methods
Analytical procedures. Protein concentrations were determined
according to the method of Lowry et a].’* Sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) was performed
according to Laemmli” on 5% to 10% acrylamide linear gradient
gels. Parallel gel lanes were stained with Coomassie Blue or
electrophoretically transferred to nitrocellulose for 3.5 hours at 240
mA and Western blotted with rabbit anti-band 3 (1:250dilution) or
rabbit anti-ankyrin (1:1,000 dilution) antiserum as described.l4.I7
Bands in the gel were quantitated by densitometric scanning, and
Western blots were similarly quantitated by scanning a photographic negative of the b10t.I~Membrane stability was examined by
ektacytometry, a laser diffraction method described
Briefly, resealed ghosts were exposed to shear stress in the
ektacytometer, and a change in their laser diffraction pattern due
to deformation of the cell was measured. The signal produced is
designated as the deformability index (DI) and is directly related to
the induced elliptical morphology of the cell. Thus, as the DI
increases, the ellipticity of the cell increases under any specific
shear stress. Measurement of this index provides an assessment of
From the Department of Chemistry, Purdue University, West Lafayette, IN; and the Department of Laboratory Medicine, Cancer
Research Institute, University of Califomia, San Francisco.
Submitted May 29, 1990; accepted November 28, 1990.
Supported in part by National Institutes of Health Grants GM24417,
DK26263, and AM16095.
Address reprint requests to Philip S. Low, PhD, Department of
Chemistry, Purdue University, West Lafayette, IN 47907-1393.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971/91/7707-OOO5$3.00/0
1581
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1582
LOW ET AL
the overall deformability of the cell. Furthermore, during constant
high-strength shearing the DI is reduced due to fragmentation of
the cell into small spherical vesicles, which are resistant to shear
deformation. Measurement of the half-time of this process provides a dependable assessment of the overall mechanical fragility of
the cell.
Analysis of polypeptide composition of Triton X-100 shells (membrane skeletons) as a function of intracellular pH. Fresh whole
blood, 50 mL, was centrifuged at 5,OOOg for 5 minutes at 4°C and
the plasma and bu@ coat were removed by aspiration. The
resulting erythrocytes were washed twice in 5 m m o w phosphate,
150 mmol/L NaCl, pH 7.4 and suspended at 50% hematocrit in this
same buffer. Diisopropylfluorophosphatewas added to 0.5 mmol/L,
and the cell suspension was incubated 30 minutes at 37°C to inhibit
endogenous proteases. Six 5-mL aliquots of the cell suspension
were then taken and pelleted by centrifuging as described above,
after which each pellet was resuspended in 40 mL of incubation
buffer (20 mmol/L Na,HPO,, 20 mmol/L N-hydroxyethylpiperazineN'-2-ethane sulfonic acid (HEPES), 20 mmol/L glycine, 100
mmoVL KCl, 5 mmol/L glucose, 2 mmol/L inosine, 150 U/mL
penicillin G potassium salt, and 0.15 mg/mL streptomycin sulfate)
at one of six pHs ranging from 7.4 to 10.4 and incubated for 15
hours at 23°C with mild agitation. Two 2-mL aliquots of the cell
suspensions were then centrifuged and analyzed to determine both
the final pH and the percent hemolysis after the overnight
incubation. The final pH was measured with a Corning 130 pH
meter directly on the previously described supernatant. The
percent hemolysis was evaluated by comparing the hemoglobin
absorbance at 562 nm of the supernatant with the hemoglobin
absorbance of an equivalent volume of nonpelleted sample treated
with 5% Triton X-100 to lyse all of the cells in the suspension. At
400 x magnification the erythrocytes appeared as biconcave discs
at all pHs up to the point of hemolysis ( pH 9.3).
The 36 mL of cell suspension not required in the above two
assays was centrifuged as described above and the cells were
resuspended in 5 mL of incubation buffer at -40% hematocrit at
the appropriate pHs. Each suspension was then lysed by adding an
equal volume of 4% Triton X-100 in incubation buffer at the same
pHs, and 9 mL of the resultant solution was layered on 35%
sucrose cushions in incubation buffer at pH 7.4 and centrifuged for
90 minutes at 200,000g. Pellets containing the Triton X-100
insoluble membrane skeletons and associated proteins (Triton
X-100 shells) were washed once by resuspending in 10 mL of 5
mmol/L phosphate, 150 mmol/L NaCl, pH 7.4 and centrifuging at
40,000g for 20 minutes. Washed pellets were then resuspended in
500 p,L of this same buffer and the protein concentration was
determined. Samples of 35 pg were subjected to SDS-PAGE and
Western blotted as described above.
Membrane stability measurements at various pHs. Washed RBCs
were lysed in 40 vol of 5 mmol/L sodium phosphate, pH 7.4,
pelleted by centrifugation at 24,000g for 5 minutes, and resuspended in 5 mmol/L NaHCO,, 1 mmoliL MgCI,, 100 mmol/L KCl,
pH 7.4. The resulting pink ghosts were then resuspended in 10 vol
of overnight incubation buffer (which had been adjusted to varying
pHs with 0.1 N NaOH) and allowed to reseal in these buffers by
incubating for 30 minutes at 37°C. The resealed ghosts were then
repelleted (24,000g for 5 minutes) and resuspended in 35%
isotonic dextran (400,000
for ektacytometric studies. The
stability of these membranes was then measured by subjecting the
membranes to a continuous shear force of 575 dynes/cm2. This
force caused the membranes to fragment with time, generating
small nondeformable spherical vesicles which led to a decrease in
the DI. The time required for the DI to decrease to 0.35 of its
original value was taken as a measure of the membrane's stability?*
-
w)
RESULTS
pH Dependence of the Composition of Triton X-100
Skeletal Shells
To study the role of the band 3-ankyrin interaction in
maintaining erythrocyte membrane stability, a method was
required that would selectively dissociate the band 3-ankyrin
linkage without significantly compromising the other essential associations in the membrane skeleton. Recent work by
Thevenin and Low'6 has demonstrated that band 3 undergoes a reversible conformational change near pH 9.2 that
converts the protein from a binding conformation (below
pH 9.2) to a form which exhibits no affinity for ankyrin
(above pH 9.2). Because pH 9.2 seemed sufficiently low to
leave most protein interactions intact, we decided to
determine whether RBCs equilibrated to pHs near 9.2
might selectively release band 3 from their membrane
skeletons without significantly jeopardizing the other major
skeletal linkages. Therefore, whole erythrocytes were incubated overnight in nutrient-containing buffers adjusted to
pHs between 7 and 10.5 and then extracted with the same
buffers containing 2% Triton X-100 (see Methods). After
sedimenting and washing the detergent-insoluble pellets in
phosphate-buffered saline, the membrane skeletal fractions
were examined by SDS-PAGE to determine the relative
retention of each major polypeptide in the membrane
skeleton. As shown in Fig lA, the content of spectrin, actin,
band 4.1, and band 4.9 remained essentially constant up to
pH 9.3, suggesting that the stability of the linkages connecting the major membrane skeletal components was not
significantly diminished by the high pH incubation. Likewise ankyrin, which because of its comigration with spectrin
had to be independently assayed by Western blotting (Fig
lC), displayed no decrease in content up through pH 9.3.
Quantitative densitometry of the above gels and blots
further confirmed the visual results (Fig 2), showing no
significant reduction in the quantity of band 4.1, actin,
spectrin, and ankyrin in the membrane skeletal complex. in
contrast, at pH 9.7 little material could be isolated in the
membrane skeletal pellet (Fig lA, lane 7), suggesting that
by this pH a major defect in the membrane skeleton had
developed leading to dissociation into protein complexes
too small to sediment through the 35% sucrose cushion.
When the behavior of band 3 as a function of pH was
subjected to similar scrutiny, a notably different titration
was observed (Fig 1, A and B). Not only did the band 3
content of the membrane skeletons begin to decrease at pH
8.4, but by pH 9.3 more than 80% of the initial skeletonassociated band 3 had been extracted. Thus, densitometric
scans of the Coomassie blue-stained gels showed a dramatic
diminution of the anion transporter in the membrane
skeletal pellet at moderate pHs, which extrapolated to near
zero levels by pH 9.5 (Fig 3). Examination of the Triton
X-100 supernatant by Western blotting with an anti-band 3
a n t i b ~ d y also
' ~ demonstrated that the soluble band 3 was
completely intact (data not shown), indicating that proteolysis was not responsible for release of the anion transporter.
Furthermore, band 4.2, an avid ligand of band 3, was also
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1583
BAND 3-ANKYRIN LINKAGE AND RBC STABILITY
A
I
B
2
3
4
5
6
7
l
2
3
4
1 2 3 4
5
6
7
ActInw
c
Fig 1. Effectof equilibrium pH
o n the composition of Triton
X-100 insoluble membrane skeletons. Erythrocytes that had been
equilibrated for 15 hours at various pHs were extracted with 2%
Triton X-100 and pelletedthrough
a 35% sucrose cushion t o remove any loosely associated proteins. The Triton-insoluble pellets were then washed and
analyzed b y SDS-PAGE and
Coornassie Blue staining (A), or
Western blotting with rabbit antiband 3 antibody (B) or rabbit
anti-ankyrin antibody (C). The
identity of the lanes is as follows: ghosts (1). Triton X-100
shells extracted from cells a t a
final equilibrium pH of 7.3 (2). 8.4
(3). 8.7 (4). 9.0 (5). 9.3 (6). and 9.7
(7). The migration positions of
major RBC membrane proteins
are indicated in the margin.
5
6
7
Ankyrin
seen to decrease Over the pH range where loss of band 3
occurred (Figs 1A and 3). Taken together, these results
indicate that by the pH where most of the skeletally
associated band 3 had been lost from the spectrin-based
skeleton, virtually none of the spectrin, actin, ankyrin, band
4.1, or band 4.9 had been released. This suggests that
equilibration of erythrocytes at pHs up to and including 9.3
might be exploited to examine the contribution of the
ankyrin-band 3 interaction to membrane fragility.
Effectof Equilibrium p H on Erythrocyte Membrane Stabilily
Two studies were conducted to evaluate the dependence
of RBC membrane stability on equilibrium pH. First,
because preparation of the aforementioned Triton X-100
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1584
LOW ET AL
I
7.5
I
8.0
85
Final pH
I
I
9.0
9.5
I
lB
.-
I
75
8.0
I
8.5
Fino1 pH
I
I
9.0
9.5
Fig 3. pH dependence of band 3 and protein 4.2 retention in Triton
X-100 shells. Data from the Coomassie Blue-stained gel in Fig 1A were
quaatitated for the presence of band 3 (0) and band 4.2 (m) by
densitometric scanning (see Methods). The results are presented as
the ratio of the amount of band 3 or band 4.2 to the amount of spectrin
in each gel lane.
v)
;t
:t
X
more characteristic of the band 3-ankyrin interaction was
obtained. Thus, ektacytometric measurements on ghosts
adjusted to pHs between 7.4 and 9.3 showed rates of
deformability index decrease that started to become significant at pH 8 and culminated in membranes unable to
withstand shear stress at pH 9.4 (Fig 5 ) . The midpoint of
this pH transition for decreased mechanical stability oc-
= 0.02
.-
E
$ 0.01
I
1
I
1
I
7.5
80
8.5
9.0
I
9.5
Final pH
I
e
Fig 2. pH dependence of the retention of membrane proteins in
Triton X-100 shells. Data from the Coomassie Blue-stained gel in Fig
1A were quantitated for the presence of actin (0)and band 4.1 (B) by
densitometry (A). Data from the Western blot of ankyrin in Fig 1C
were quantitated as described in Methods (B). Results for ankyrin are
reported as the peak area (measured in grams) of the densitometric
scan of the ankyrin band in the blot.
shells required overnight incubation of intact erythrocytes
at elevated pH, we decided to examine the same RBC
suspensions to determine whether equilibration at any of
the pHs might lead to unstimulated hemolysis. For this
purpose, the free hemoglobin content of the extracellular
medium was assayed after overnight incubation and compared with the total hemoglobin content of each suspension. As shown in Fig 4, no significant hemolysis was seen in
these “mechanically unstressed” cells until pH 9.2, where
released hemoglobin began to increase exponentially. Based
on a visual comparison with the extraction studies of Figs 1
and 3, it was concluded that the pH dependence of
unstressed hemolysis correlated better with the pH dependence of membrane skeletal fragmentation than with the
pH dependence of the band 3-ankyrin interaction, ie,
measurable fragmentation was observed only above pH 9.3
(Fig lA, lane 7).
In contrast, when resealed membranes equilibrated to
the same pHs were subjected to mechanical stress, a
pH-dependent decrease in membrane mechanical stability
-
Final pH
Fig 4. RBC hemolysis as a function of equilibrium pH. Erythrocytes
were incubated for 15 hours in incubation buffer (see Methods). The
cells were then pelleted and the pHs of the supernatants were
determined. This is reported as the final pH. The hemoglobin concentration of the same supernatant was determined from the absorbance
at 562 nm, and the percent hemolysis was calculated by dividing this
value by the hemoglobin concentration of a parallel sample of cells
that were 100% hemolyzed with 5% Triton X-100 (see Methods). At
the higher pH values, the final pH after equilibration was 0.5 t o 1.0 pH
U less than the pH of the initial suspending buffer.
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1585
BAND 3-ANKYRIN LINKAGE AND RBC STABILITY
I
I
I
50
I
100
I
150
200
time ( s e d
75
80
8.5
9.0
9.5
PH
Fig 5. pH dependence of membrane stability in resealed ghosts.
The deformability index of resealed ghosts, prepared at the pHs
indicated, was measured in the ektacytometer as described in Methods. (A) DI tracings with time at various pH values from one representative experiment. (B) A plot of the average of the DI data from three
different experiments. The percent of the pH 7.4 control was calculated from the ratio of the time required to decrease to DI 0.35 at a
given pH versus the time to DI = 0.35 at pH 7.4 (control). Standard
deviations between experiments varied between 9.3% at pH 8.1 to
4.9% at pH 9.3.
-
curred at pH 8.7, slightly below the mid pH of the band 3
dissociation transition (pH 8.8, Fig 3), but well within the
range of experimental variability. Importantly, the pH
dependence of lzI-labeled ankyrin binding to inside-out
erythrocyte membrane vesicles after a 26-hour incubation
also yielded a pH dependence similar to the membrane
mechanical stability changes, but vastly distinct from the
unstimulated hemolysis curve.I6 Thus, one hypothesis to
explain the apparent correlation between band 3 extractability and membrane stability is that the band 3-ankyrin
interaction is intrinsically involved in protecting the cell
against fragmentation by mechanical stress, but not essential in preventing the hemolysis of mechanically unstressed
cells that occurs at higher pH.
DISCUSSION
We have presented evidence that the predominant linkage of the lipid bilayer to the membrane skeleton can be
gradually dissociated at pHs where the other major membrane skeletal interactions remain largely intact. Although
both band 3 and band 4.2 are released from the membrane
skeletons over this same pH range, we interpret the effect
of pH on the release of band 3 to be due directly to
dissociation of the band 3-ankyrin complex and not a
consequence of the titration of some band 4.2 interaction.
Our reasons for this contention are as follows. First, the
dissociation of band 3 from the membrane skeleton closely
followed the pH dependence of the band 3-ankyrin association at equilibrium in vitroI6 and was therefore predicted
from these model studies. Second, band 3 binds ankyrin
(K, = lo-* molk) independently of band 4.2 and, therefore, it is unlikely that loss of band 4.2 from the membrane
skeleton could promote dissociation of band 3.7*8.’4.‘6*23-25
In
this respect it should be noted that erythrocytes from a
patient completely lacking band 4.2 have been found
recently to exhibit an apparently normal band 3-ankyrin
interaction and near-normal membrane mechanical stability.” Finally, the co-extraction of band 4.2 with band 3 as
pH was elevated was anticipated from the known association of band 4.2 with band 3.26Thus, as band 3 is released
from its pH-sensitive interaction with ankyrin, the tightly
associated band 4.2 would also be expected to extract.
Although no disappearance of spectrin, actin, or band 4.1
from the membrane skeletons was observed below pH 9.3,
the complete loss of membrane mechanical stability before
pH 9.3 (Fig 5B) could have been influenced by factors other
than the pH-dependent dissociation of band 3. Thus,
interactions among the skeletal components could have
weakened without leading to fragmentation of any skeletal
linkage. Also, dissociation of the band 3-ankyrin complex
could have encouraged some spectrin dissociation via a
positive cooperativity reported to exist in the band 3-ankyrinspectrin li11kage.2~Nevertheless, based on the strong correlation between dissociation of the band 3-ankyrin linkage
and the membrane fragmentation pattern under shear
stress, we suggest that the dominant defect responsible for
membrane instability between pH 8 and 9.2 is the progressive dissociation of ankyrin from band 3.
The hypothesis that a strong membrane skeleton-bilayer
junction may be essential for cell survival under mechanical
deformation, but not for the maintenance of membrane
integrity in the absence of shear stress, obviously requires
further investigation. However, even in the absence of
additional documentation, the proposal can still be argued
largely on theoretical grounds. Thus, when two metastable
layers are bonded together at regular intervals, a process
termed “load transfer” can occur where a stress normally
assumed by one of the layers is shared between both
layers.” Under these conditions a mechanical distortion or
wave that might normally fragment one of the layers would
be rapidly attenuated in the linked system. By analogy,
whereas the membrane skeleton alone may be sufficient to
maintain membrane integrity in the absence of shear stress,
multiple junctions to the adjacent bilayer may be necessary
to resist hemolysis in the face of mechanical deformation.
Therefore, the significance of the band 3-ankyrin linkage
may be to protect the RBC against hemolysis during tight
passage through the narrow capillaries of the circulatory
system.
In this regard it is interesting to note that absence of
another linkage between the bilayer and the membrane
skeleton (that of glycophorin C with protein 4.1) has also
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LOW ET AL
1586
been shown to result in decreased mechanical ~tability.2~
The present data, in conjunction with this finding, imply
that vertical linkages between the bilayer and skeletal
complex are crucial for maintaining the mechanical integrity of the erythrocyte membrane. While much of the
attention to date has been focused on spectrin-spectrin
self-association and spectrin-actin-protein 4.1 interaction
in regulating the structural integrity of the membrane, our
data suggest that the band 3-ankyrin interaction may also
play a crucial regulatory role.
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1991 77: 1581-1586
Contribution of the band 3-ankyrin interaction to erythrocyte
membrane mechanical stability
PS Low, BM Willardson, N Mohandas, M Rossi and S Shohet
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