Báo cáo khoa học: The molecular chaperone a-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress ppt

The molecular chaperone a-crystallin incorporated into red cell
ghosts protects membrane Na/K-ATPase against glycation
and oxidative stress
Barry K. Derham
1
, J. Clive Ellory
2
, Anthony J. Bron
1
and John J. Harding
1
1
Nuffield Laboratory of Ophthalmology, University of Oxford, UK;
2
Laboratory of Physiology, University of Oxford, UK
a-Crystallin, a molecular chaperone and lens structural
protein protects soluble enzymes against heat-induced
aggregation and inactivation by a variety of molecules. In
this study we investigated the chaperone function of a-crys-
tallin in a more physiological system in which a-crystallin was
incorporated into red cell ÔghostsÕ. Its ability to protect the
intrinsic membrane protein Na/K-ATPase from external
stresses was studied. Red cell ghosts were created by lysing
the red cells and removing cytoplasmic contents by size-
exclusion chromatography. The resulting ghost cells retain
Na/K-ATPase activity. a-Crystallin was incorporated in the
cells on resealing and the activity of Na/K-ATPase assessed
by ouabain-sensitive
86
Rb uptake. Incubation with fructose,
hydrogen peroxide and methylglyoxal (compounds that have

been implicated in diabetes and cataract formation) were
used to test inactivation of the Na/K pump. Intracellular
a-crystallin protected against the decrease in ouabain sensi-
tive
86
Rb uptake, and therefore against inactivation induced
by all external modifiers, in a dose-dependent manner.
Keywords: a-crystallin; ghost cells; glycation; Na/K-ATPase;
oxidation.
Na/K-ATPase is a highly conserved, ubiquitous membrane
protein. The enzyme is composed of three subunits; the
alpha subunit ( 113 kDa) binds ATP and sodium and
potassium ions, and contains the phosphorylation site. The
smaller beta subunit ( 35 kDa glycoprotein) is necessary
for activity of the complex and the gamma subunit
( 10 kDa) is involved with modulation of Na/K-ATPase.
Several isoforms of both alpha and beta subunits have
been identified [1].
Red blood cell membranes contain Na/K-ATPase.
Removal of the erythrocyte cytoplasm by lysis followed
by size-exclusion chromatography produces white ghost
cells showing Na/K-ATPase activity. Erythrocyte ghost
Na/K-ATPase activity can be determined by measuring
the ouabain-sensitive uptake of
86
Rb (as a congener for
potassium). Tissue proteins existing within an environment
containing reactive small molecules such as sugars, cyanate,
methylglyoxal and other reactive metabolites are vulnerable
to nonenzymatic modification that may affect their physio-

logical function [2]. Such post-translational modifications
contribute to systemic and ocular disease including cataract
and the complications of diabetes.
Glycation, a process that is pertinent to the aetiology of
diabetes, is initiated by the reaction between the carbonyl
group of a sugar with an amino group (usually a lysine or
the N-terminal amino group) of a protein, to form a Schiff
base. This may undergo a further Amadori rearrangement,
to produce a ketoamine. There is evidence from experimen-
tal diabetes that glycation may play a central role in the
impairment of Na/K-ATPase activity in this disorder and
contribute to the pathophysiology of diabetic complications
[3].
Glycation has far-reaching consequences including the
production of increased amounts of the reactive metabolite
methylglyoxal, especially in the lens [4]. Methylglyoxal is a
reactive a-dicarbonyl with 100% open chain that modifies
proteins more rapidly than glucose by an interaction with
arginine and cysteine, in addition to lysine [2]. It has been
shown to cross-link proteins during glycation or Maillard
reactions resulting in protein-bound fluorescent molecules
or advanced glycation end products [5]. At physiological
concentration (1 l
M
) methylglyoxal binds to proteins in
blood plasma [6].
Reactive oxygen species such as hydrogen peroxide
(H
2
O

2
) are continually produced in biological systems as
unwanted by-products of normal oxidative metabolism.
Antioxidant defences detoxify these reactive oxygen species,
but increased production by various biological and envi-
ronmental factors can lead to oxidative damage to key
molecules such as lipid, protein, DNA, etc.
Previous experiments in our laboratory have demon-
strated inactivation of enzymes by fructose, cyanate
and prednisolone-21-hemisuccinate. Fructation causes a
decrease in activity of a range of enzymes in vitro [7–9], and
the inactivation was prevented by a-crystallin. a-Crystallin,
a lens structural protein, comprising of aAandaB subunits
is a ubiquitous molecular chaperone, which has been
shown to protect many enzymes from inactivation and
heat-induced aggregation [10]. Ingolia and Craig [11]
discovered an approximate 55% sequence homology
Correspondence to J. J. Harding, Nuffield Laboratory of Ophthal-
mology, University of Oxford, Walton Street, Oxford, OX2 6AW,
UK. Fax: + 44 1865 794508, Tel.: + 44 1865 248996,
E-mail:
Enzymes: creatine kinase (EC 2.7.3.2, type 1 from rabbit muscle).
(Received 10 February 2003, revised 7 April 2003,
accepted 23 April 2003)
Eur. J. Biochem. 270, 2605–2611 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03631.x
between small heat shock proteins from Drosophila
melanogaster and bovine a-crystallin. Horwitz [12] first
characterized a-crystallin as a molecular chaperone in vitro,
based on its ability to prevent heat-induced aggregation
of lens proteins and enzymes. These protective capabilities

have been demonstrated with other, in vitro systems,
including prevention of aggregation of insulin B chain
following reduction of disulphide bonds [13], refolding of
guanidine hydrochloride (or urea)-denatured proteins
[12,14] and prevention of inactivation of enzymes by small
molecules [7,8]. a-Crystallin has also been shown to
decrease the degree of thiol oxidation of other lens crystal-
lins under conditions of oxidative stress [15]. Characteri-
zation of a-crystallin using these assays has indicated
similar mechanisms of protection. However, the molecular
mechanism of the interaction between a-crystallin and
substrates remains enigmatic. Recently, we have shown
that a-crystallin incorporated into ghost cells protects
soluble enzymes such as catalase, malate dehydrogenase
and glutathione reductase from inactivation by fructose
[16]. Enzymes were resealed within ghost cells and inacti-
vated by fructose. When a-crystallin was resealed with the
enzyme, activity was retained.
In the present study we demonstrate the protection of
the membrane enzyme Na/K-ATPase from inactivation
by the heat shock protein a-crystallin. Na/K-ATPase
activity decreased upon incubation with fructose, H
2
O
2
and methylglyoxal. However, Na/K-ATPase activity was
preserved when the heat shock protein a-crystallin was
sealed within the ghost cells. In this situation a-crystallin
was able to protect against each form of modification.
Methods

Materials
86
Rb was purchased from NEN Life Sciences. All other
chemicals and enzymes, including luciferin–luciferase firefly
lantern extracts were obtained from Sigma. Sepharose 2B
and Sephacryl S300 H were obtained from Pharmacia Ltd.
a-Crystallin was isolated from bovine lenses by Sephacryl
S300 H size-exclusion chromatography as described by
Derham and Harding [17].
Preparation of the ghost cells
Freshly drawn human blood (30 mL) was collected from
volunteers with consent and stored at 4 °C with heparin for
a maximum of 3 days. Erythrocyte ghosts, free of haemo-
globin were prepared by gel filtration chromatography [18].
Blood (5–6 mL) was centrifuged (1000 g) for 10 min, the
plasma and white cells aspirated and the red cells resus-
pended at 0 °C with isotonic Hepes buffer (20 m
M
Hepes,
146 m
M
NaCl, pH 7.4). This procedure was repeated four
times. After the final wash the supernatant was aspirated
and the packed red cells ( 3 mL) were lysed with
hypotonic buffer (15 m
M
Pipes, 0.1 m
M
EDTA pH 6, and
approx. 50 mOsm) at a 10% haematocrit. The suspension

was shaken gently and cooled in an ice bath for 5 min
before loading onto a Sepharose 2B size-exclusion column
(5 · 28 cm) pre-equilibrated with the hypotonic Pipes
buffer and maintained at 0 °C by a cooling jacket with
circulating antifreeze. The column was eluted with Hepes
buffer at a constant flow rate of 30 mLÆh
)1
and fractions
collected in tubes in an ice bath to prevent resealing. The
ghost cells eluted in the void volume (70 mL) while the main
haemoglobin band followed about 130 mL later (Fig. 1).
The lysed cells were white and therefore practically haemo-
globin-free. The lysed cells were collected by centrifugation
(11 000 g,10min,0°C), the supernatant aspirated and
the pellet re-suspended in isotonic Hepes buffer at 0 °Cto
prevent resealing. This washing procedure was repeated
four times, to remove the hypotonic buffer. The low
temperature prevents the ghost cells resealing.
Re-sealing ghost cells
After the final wash the supernatant was aspirated and the
packed ghost cells were suspended in 5 mL of resealing
buffer at 0 °C. Resealing buffer contained NaCl (10 m
M
),
KCl (140 m
M
), Mops (10 m
M
), dithiothreitol (2 m
M

),
EGTA (0.1 m
M
), potassium phosphate (1 m
M
)andMgCl
2
(0.15 m
M
) at pH 7.4. Potassium ATP (2 m
M
), sodium
phosphocreatine (5 m
M
) and creatine kinase (EC 2.7.3.2,
type 1 from rabbit muscle) 5 UÆlL
)1
were added as an
ATP-regenerating system to maintain membrane integrity
[19].
The lysed cell suspension was divided equally into two
tubes and to one tube a-crystallin (1 mgÆmL
)1
) was added
and incorporated on resealing. For controls, BSA
(1 mgÆmL
)1
) and lysozyme (1 mgÆmL
)1
)wereusedinstead

of a-crystallin.
The tubes containing the suspensions were placed on ice
for 10 min then at 37 °C for 30 min so that the lysed ghost
cells would reseal. After resealing the ghost cells were
washed three times in Mops buffer (10 m
M
Mops, 146 m
M
sodium nitrate) and successively centrifuged (10 000 g,
5 min) and re-suspended to achieve chloride replacement
(to eliminate K-Cl cotransporter activity [20,21] and remove
resealing solution.
Fig. 1. Elution profile of a haemolysed erythrocyte suspension loaded
onto a Sepharose 2B size-exclusion column (5 cm · 28 cm) pre-equili-
brated with hypotonic Pipes buffer pH 6 that was maintained at 0 °C.
The void volume at  70 mL contains the ghosts. Haemoglobin eluted
at 200 mL.
2606 B. K. Derham et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Measurement of
86
Rb flux while modifying Na/K-ATPase
The activity of Na/K-ATPase was assessed by ouabain-
sensitive
86
Rb uptake. Ghost cells were incubated at 37 °C
in Mops buffer and assayed at time zero and after 6 h.
Resealed ghost cells, with and without incorporated
a-crystallin, were suspended in Mops buffer to give a final
haematocrit of  10%. Resealed cells were aliquotted into
1.5-mL Eppendorf tubes (triplicate) and were assigned to

the addition of: (a) buffer (control); (b) ouabain (0.1 m
M
);
(c) modifier; and (d) modifier and ouabain. The modifiers
included sucrose (50 m
M
), fructose (50 m
M
), methylglyoxal
(0.1 m
M
,1m
M
and 10 m
M
)andH
2
O
2
(0.5 m
M
). All
experiments were performed in triplicate.
The Na/K-ATPase flux was initiated by the addition
86
Rb and sufficient potassium nitrate to yield a final
concentration of 7.5 m
M
(20 lCi
86

Rb per ml buffer). The
tubes were shaken and then incubated at 37 °C for 30 min,
transferred onto ice for 1.5 min, centrifuged (2 min,
10 000 g), and the supernatant removed by aspiration.
The ghost cells were washed free of
86
Rb by four successive
re-suspensions and centrifugation in ice-cold wash solution
(0.1
M
MgCl
2
,10m
M
Mops pH 7.4). The cell pellet was
lysed by addition of 0.5 mL Triton 0.1% (v/v) (TX-100),
precipitated by the addition of 0.5 mL 5% w/v trichloro-
acetic acid and centrifuged (5 min, 10 000 g). The super-
natant was transferred into scintillation vials for Cerenkov
counting in a b-scintillation spectrometer. Flux was
expressed as percentage control (mean of a minimum of
five experiments). The data were analysed using a Student’s
paired t-test, with P <0.05 (*), P < 0.01 (**) and
P < 0.001 (***) considered statistically significant.
ATP determination
ATP levels were measured using a luciferin–luciferase
enzyme system [22] using a BioOrbit 1253 luminometer.
One vial of freeze-dried firefly lantern extract was reconsti-
tuted with 5 mL distilled water 1–2 h before assay and
stored on ice. To 1 mL buffer (100 m

M
Tris/HCl pH 6.8,
5m
M
MgSO
4
,0.5m
M
EDTA, 0.5 m
M
dithiothreitol,
0.1 mgÆmL
)1
human serum albumin) was added 50 lL
luciferin–luciferase solution and 10 lL sample and gently
stirred. After 30 s the light emitted from the ATP-dependent
firefly extract was determined and result subtracted from the
background value. A calibration curve was set up. All
readings were in triplicate.
Na and K photometry determination
An IL943 flame photometer (Instrumentation Laboratory,
Lexington, MA, USA) was used for the determination of
sodium and potassium within the erythrocyte ghost cells.
Values of Na
+
and K
+
were measured and expressed in
m
M

ÆL
)1
. All readings were taken in triplicate.
SDS/PAGE
SDS/PAGE (12.5% w/v gel) was performed as described by
Laemmli [23] under reducing conditions with a Bio-Rad
system. Ghost cells (20 lL) were dissolved in the sample
buffer containing 5% (v/v) 2-mercaptoethanol. Coomassie
brilliant blue G was used to detect the polypeptide bands.
The relative abundance of sample band was determined by
quantitative analysis of digital photographs of gels on a
computer (Labworks, UVP Products, Upland, CA, USA).
Resealing assay
To check that ghosts sealed tightly in our procedure,
14
C
sucrose (50 lCiÆmL
)1
) (unlabelled sucrose acted as control)
was resealed inside control ghost cells. After resealing the
ghost cells were sequentially washed three times in Mops
buffer (10 m
M
Mops, 146 m
M
sodium nitrate) and centri-
fuged (10 000 g, 5 min). The ghost cells were then incubated
for 2 h at 37 °C. Samples from the ghost cells and the
supernatants were taken for scintillation counting.
Creatine kinase determination

Creatine kinase activity was assayed following the method
of Bernt and Bergmeyer [24], in the presence of fructose
(50 m
M
) to determine if the inhibitory effect of the modifier
was through inhibition of the ATP-regenerating system.
Results
The ghost cells eluted through the Sepharose 2B size
exclusion column in the void volume while haemoglobin
and cellular enzymes eluted distinctly later (Fig. 1), com-
parable to a previously published gel filtration method of
ghost cell preparation [18]. The eluted ghost cells are in their
open form, allowing various proteins to be incorporated
before resealing.
SDS/PAGE analyses of ghost cells alone showed the
usual pattern for red cell membrane proteins (Fig. 2, lane 2).
Added a-crystallin ( 800 kDa) was incorporated inside
the ghost cells. Ghosts resealed in the presence of a-
crystallin showed the extra two a-crystallin subunit bands at
around 20 kDa confirming the efficient sealing of the
protein within the cells (Fig. 2, lane 3). a-Crystallin alone
showed clear bands at the expected subunit weight of
20 kDa (lane 4). a-Crystallin was loaded at 5 mgÆmL
)1
onto
theSDS/PAGEtohighlightthefactthatitcanberesealed,
and at high concentrations. These results are in accordance
with previous resealing experiments [16]. BSA reseals inside
the ghost cells with equal efficiency (result not shown).
To confirm that the lysing and resealing procedure

produced effectively sealed ghost cells,
14
C sucrose was
resealed into ghost cells. The cells were washed three times
to remove radioactivity in the supernatant and incubated
for 2 h at 37 °C. After this time no radioactivity was
detectable in the supernatant. At the same time, significant
radioactivity of
14
C sucrose in the ghost cells was measured
whichwasthesameatt ¼ 0 and 2 h. This indicates that the
14
C sucrose was trapped by loading and resealing and did
not leak out of the ghost cells. The ghost cells were
incubated with and without modifiers over 6 h at 37 °C,
after which time the activity of Na/K-ATPase was measured
over a 30-min period by
86
Rb uptake. A steady-state of Na,
86
Rb exchange is achieved during a 30 min assay. The
concentration levels of the modifiers were selected so that
they would not interfere with the chaperone function of
a-crystallin reported previously [10].
Ó FEBS 2003 a-Crystallin protects Na/K-ATPase in red cell ghosts (Eur. J. Biochem. 270) 2607
The effects of ouabain, a-crystallin and sugars on the
86
Rb uptake of the ghost cells are shown in Fig. 3. Ouabain
(0.1 m
M

), a specific inhibitor of Na/K-ATPase, caused
a 35% decrease in the
86
Rb uptake within the ghost cells.
This demonstrates that the ghosts have functional Na/K-
ATPase. The presence of a-crystallin within the ghost cell
did not cause any decrease in
86
Rb uptake. The presence of
50 m
M
sucrose, a nonreducing sugar, on
86
Rb uptake after a
6 h incubation caused no significant decrease in
86
Rb
uptake.
When the ghost cells were incubated with 50 m
M
fructose
for 6 h the
86
Rb uptake was inhibited by  45%, which is
 10% greater than that induced by ouabain. When the
experiment was repeated with ouabain and 50 m
M
fructose,
the
86

Rb uptake was inhibited by the same amount as before
(fructose alone). This suggests that fructose inhibited all the
Na/K-ATPase activity. When the ghost cells were incubated
with 50 m
M
fructose for 6 h with a-crystallin (1 mgÆmL
)1
)
resealed inside the ghost cells, the
86
Rb uptake was
maintained at  90% of control (P < 0.001 compared to
inhibition by fructose).
To show that the protection that a-crystallin provided
against Na/K-ATPase inactivation was not due to the
removal of fructose by binding to a-crystallin, BSA was
resealed (in the same manner and concentration as that of
a-crystallin) and incubated with 50 m
M
fructose for 6 h.
BSA (1 mgÆmL
)1
) was used as a control protein because it
has a greater lysine content than a-crystallin and therefore a
greater ability to bind fructose. The resealed BSA did not
display any protective activity and the
86
Rb uptake was
similar to that with fructose alone, i.e.  45% inhibition
(Fig. 3).

When the ghost cells were incubated with 0.1 m
M
methylglyoxal for 6 h the
86
Rb uptake was inhibited by
 20% (Fig. 4). When the experiment was repeated but
Fig. 2. SDS/PAGE of red cell ghosts with and without a-crystallin
resealed. Lanes 1 and 5, molecular mass markers; lane 2, ghost cells
alone; lane 3, ghost cells resealed with a-crystallin present, 5 mgÆmL
)1
(the double band around 20 kDa indicates presence of a-crystallin);
lane 4, a-crystallin alone, 5 mgÆmL
)1
(double band around 20 kDa).
Fig. 3. Effects of 0.1 m
M
ouabain, 50 m
M
sucrose and 50 m
M
fructose
on the
86
Rb uptake into red cell ghosts after 6 h incubation at 37 °C, and
the effect of the molecular chaperone a-crystallin (1 mgÆmL
-1
)andBSA
(1mgÆmL
-1
) separately resealed inside red cell ghosts upon the rubidium

uptake of those modifiers. Error bars represent standard deviation.
Fig. 4. Effects of 0.1, 1 and 10 m
M
methylglyoxal and 0.1 m
M
ouabain
on the
86
Rb uptake into red cell ghosts after 6 h incubation at 37 °C, and
the effect of the molecular chaperone a-crystallin (1 mgÆmL
-1
) resealed
inside red cell ghosts upon the rubidium uptake of those modifiers. Error
bars represent standard deviation.
2608 B. K. Derham et al. (Eur. J. Biochem. 270) Ó FEBS 2003
with the addition of ouabain, the
86
Rb uptake was inhibited
by  35%, indicating that 0.1 m
M
methylglyoxal did not
inhibit ouabain-sensitive
86
Rb uptake completely. When the
ghost cells were incubated with 0.1 m
M
methylglyoxal for
6hwitha-crystallin (1 mgÆmL
)1
) resealed inside the ghost

cells, the
86
Rb uptake was maintained at 100% of control
(P < 0.05 compared to inhibition by methylglyoxal).
Increasing concentrations of methylglyoxal caused a dose-
dependent decrease in
86
Rb uptake. Incubation with 1 m
M
methylglyoxal inhibited
86
Rb uptake by  40%, the pres-
ence of ouabain however, did not change the amount of
inhibition suggesting that most of the Na/K-ATPase had
been modified. When a-crystallin (1 mgÆmL
)1
) was resealed
inside the ghost cells that were incubated with 1 m
M
methylglyoxal
86
Rb uptake was restored to  90% of the
control (P < 0.05 compared to inhibition by methylgly-
oxal). At 10 m
M
methylglyoxal the
86
Rb uptake of the ghost
cells was inhibited by  50%, and the presence of ouabain
did not inhibit it further. This suggests that 10 m

M
methylglyoxal was inhibiting other K
+
permeability path-
ways, in addition to Na/K-ATPase. When the experiment
was repeated with a-crystallin (1 mgÆmL
)1
) resealed on the
inside
86
Rb uptake was maintained at  85% of the control
values (P < 0.01 compared to inhibition by methylglyoxal
alone).
Ghost cells were subjected to oxidative stress in the form
of H
2
O
2
. When the ghost cells were incubated with 0.5 m
M
H
2
O
2
for 6 h the
86
Rb uptake was inhibited by  40%, the
additional presence of ouabain did not change the degree of
inhibition significantly (Fig. 5). When the ghost cells were
incubated with 0.5 m

M
H
2
O
2
for 6 h with a-crystallin
(1 mgÆmL
)1
) resealed inside the ghost cells, the
86
Rb uptake
was restored to  85% of control (P < 0.01 compared to
inhibition by H
2
O
2
). When the ghost cells were incubated
with 0.5 m
M
H
2
O
2
, ouabain and a-crystallin, the
86
Rb
uptake was approximately the same, as that with ouabain
alone, indicating a selective effect of H
2
O

2
on the Na, K
pump.
To ensure that changes in
86
Rb flux were not due to
alterations in ATP concentrations or Na
+
and K
+
levels
three control experiments were performed.
The efficiency of the ATP regenerating system was
checked: ATP levels over 6 h were measured, as were
concentrations of Na
+
and K
+
. Without the ATP regen-
erating system active transport via Na/K-ATPase measured
as
86
Rb flux is greatly reduced (results not shown). The
activity of creatine kinase did not decrease after 6 h
incubation with fructose (results not shown).
ATP levels within resealed ghost cells at time zero and at
6 h incubations were measured using a luciferin–luciferase
enzyme system (Fig. 6). Ghost cells were prepared and
incubated with modifiers as described. The ATP levels
within the treated ghost cells at time zero were approxi-

mately equal to those of the control ghost cells. The control
value at 6 h had decreased by  30%, and the modified
ghost cells showed similar decreases in ATP levels (Fig. 6).
Thus, the differences in
86
Rb flux are not caused by a
lowering of ATP.
Fig. 5. Effects of 0.5 m
M
H
2
O
2
and 0.1 m
M
ouabain on the
86
Rb uptake
into red cell ghosts after 6 h incubation at 37 °C, and the effect of the
molecular chaperone a-crystallin (1 mgÆmL
-1
) resealed inside red cell
ghosts upon the rubidium uptake of those modifiers. Error bars represent
standard deviation.
Fig. 6. ATP levels in ghost cells subjected to various challenges. ATP
levels were measured using a luciferin–luciferase enzyme system using a
BioOrbit 1253 luminometer. Ghost cells were prepared as described
in the methods section and incubated at 37 °C, 10 lLsamplestaken
at t ¼ 0andt ¼ 6 h and assayed. Error bars represent standard
deviation.

Ó FEBS 2003 a-Crystallin protects Na/K-ATPase in red cell ghosts (Eur. J. Biochem. 270) 2609
Flame photometry was used for determining the levels of
sodium and K
+
within the ghost cells at time zero and 6 h.
At time zero the control ghost cells had a Na
+
concentra-
tion of approximately 110 m
M
, which did not decrease over
6 h. All modifiers had zero time Na
+
concentrations of
 100 m
M
that did not decrease over 6 h. The K
+
concentration in all ghost cells, control and modified, were
 13 m
M
at time zero and  9m
M
at time 6 h. Thus the
changes in
86
Rb flux were not due to changes in Na
+
or
K

+
.
Discussion
The membrane protein Na/K-ATPase of red blood cell
ghosts was stressed using externally applied fructose,
methylglyoxal and H
2
O
2
and activity measured by
86
Rb
uptake. We report for the first time the ability of the
chaperone protein a-crystallin, to prevent the inhibition of
the membrane-bound enzyme by fructose, methylglyoxal
and H
2
O
2
. a-Crystallin was able to protect Na/K-ATPase
from inactivation by all the modifiers. a-Crystallin has
previously been shown to protect soluble unfolding proteins
by forming stable high molecular weight complexes that
retain their functional state, but does not refold the proteins
back into the native state [7,8,17]. This study implies that
a-crystallin protects Na/K-ATPase in a similar manner
from the cytosolic side of the ghost cell. The means by which
it affords such protection is unknown but presumably
inactivation of the enzyme by the modifier is due to the
targeting of a domain of the enzyme that is accessible to

both the modifier and to intracellular a-crystallin. The
cytoplasmic loop of Na/K-ATPase might provide such a
target for protection. This would be in keeping with the
failure of a-crystallin to reverse ouabain-induced inhibition
of the enzyme. It is thought that a-crystallin may act
through dynamic interactions, such that the chaperone may
prevent further unfolding but not bind to the target protein.
Binding of a-crystallin to ghost cell membranes was not seen
under experimental conditions (results not shown). This has
been previously observed by [25] looking at soluble proteins.
It is possible that more severe conditions are necessary for
complex formation.
The process of ghost cell preparation from red blood cells
by lysis followed by size exclusion chromatography pro-
duced very pure intact membranes with fully operational
ion transporters and an intact cytoskeleton [18]. Production
of red blood cell ghosts by hypotonic lysis results in the
formation of a large number of pores in the red cell
membrane, estimated to be 30 nm in diameter [26].
a-Crystallin was shown to reseal within ghosts (Fig. 2).
Other molecules that have been resealed include albumin
(70 kDa) [27], ferritin (474 kDa; diameter 8 nm) and gold
particles (10–15 nm) [26]. The amounts of a-crystallin
to Na/K-ATPase were determined by densitometry of a
SDS/PAGE gel (a-crystallin 1 mgÆmL
)1
resealed inside a
ghost cells), using the b-subunit (35 kDa) of the Na/K-
ATPase as reference. The ratio of a-crystallin to the
b-subunit was  2 : 1 by mass, a ratio previously observed

between target enzymes and a-crystallin when a-crystallin
protected the enzyme activity [9].
The decreases in
86
Rb flux caused by fructose, methyl-
glyoxal and H
2
O
2
were not caused by impairment of the
ATP regeneration system, nor by loss of ATP or by changes
in concentrations of Na
+
and K
+
levels. These experiments
were performed in the absence of a-crystallin, suggesting
that the chaperone function of a-crystallin is committed to
protecting primarily membrane-bound proteins.
The uptake of
86
Rb in the ghost cells with ouabain
(0.1 m
M
) decreased by  35%. This is the amount of
86
Rb
flux across the ghost cell membrane contributed by Na/K-
ATPase. The other 65% of K influx presumably reflects an
increased K leak pathway in the resealed ghosts.

The protection is not a result of a-crystallin reacting with
free inactivators as a-crystallin does not simply compete for
fructose [7]. Incorporation of radiolabelled fructose with
proteins such as lysozyme, which has a similar lysine
content, and BSA, which has a greater lysine content, all
bind fructose at a similar rate and displayed no chaperone
protective ability [7]. Previous experiments have demon-
strated that increased incorporation of radiolabelled fruc-
tose mirrored a decline in activity of glucose-6-phosphate
dehydrogenase [8]. Protection may be via transient dynamic
complex formation that would allow enzymes, soluble and
membrane bound, to retain their functional state.
All modifiers studied here can cross the ghost cell
membrane easily; they diffuse through the membrane
because they are not charged. There is no active transport
for methylglyoxal or H
2
O
2
in the erythrocyte membrane.
There is an active glucose-transporter but it is specific for
glucose and not for other hexoses; fructose can move
through but 15 times slower then glucose. It is thought that
this would not be significant to the overall amount of
fructose in the cell. The modifiers can all pass through the
membrane so a steady state would be achieved at the
molarity of the modifying agent.
The site of modification differs slightly between modifiers.
Fructose reacts with lysine residues, whereas methylglyoxal
reacts principally with arginine residues although modifica-

tion of lysine and cysteine also occurs [2,28]. H
2
O
2
oxidizes
methionine and cysteine residues as well as lipids [29].
Effective defence systems exist intracellularly to reduce these
modifications such as catalase, glutathione peroxidase and
glutathione. The reduced glutathione-dependent glyoxalase
converts methylglyoxal to
D
-lactate [30]. Abnormalities in
Na/K-ATPase activity are thought to be involved in several
pathologic states, in particular heart disease, hypertension
and cataract. Altered Na
+
and K
+
concentrations are
observed in many forms of human cataract and correlate
with increasing lens colour and with cortical opacification
[31]. The change in monovalent cation concentrations
may in part be attributed to decreased efficiency of the
Na/K-ATPase.
Resealing of the molecular chaperone a-crystallin within
a red cell ghost, followed by stress from post-translational
modifications protected the ghost cell Na/K-ATPase. This
type of assay provides additional evidence of the important
role of the small heat shock proteins in cell protection. Also,
the protection from modification of ghost cell Na/K-

ATPase by a-crystallin highlights the diverse nature of
molecular chaperones and suggests that protection of
Na/K-ATPase is ubiquitous to all cells, not just red cell
membranes. Heat stress in NIH3T3 cells causes a transient
decrease of aB-crystallin levels from the cytosol as it is
translocated reversibly to the membrane providing protein
2610 B. K. Derham et al. (Eur. J. Biochem. 270) Ó FEBS 2003
synthesis is not inhibited [32]. Heat shock of Reuber H35
hepatoma cells did not cause decrease in ouabain-sensitive
86
Rb influx [33], possibly because of transient protection
from heat shock proteins.
As far as we are aware this is the first report of
the protection of a membrane enzyme by a molecular
chaperone.
Acknowledgements
We are grateful to the Wellcome Trust and to the Knoop Trust for a
Junior Research Fellowship. We are grateful to Dr Steve Ashcroft, at
the Diabetes Research Laboratories, University of Oxford for the use of
his luminometer; and to Dr Simon Golding at the Department of
Physiology, University of Oxford for the use of the flame photometer.
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Ó FEBS 2003 a-Crystallin protects Na/K-ATPase in red cell ghosts (Eur. J. Biochem. 270) 2611