Resealing assay To check that ghosts sealed tightly in our procedure, 14C sucrose 50 lCiÆmL1 unlabelled sucrose acted as control was resealed inside control ghost cells.. The ghost cells
Trang 1The molecular chaperone a-crystallin incorporated into red cell
ghosts protects membrane Na/K-ATPase against glycation
and oxidative stress
Barry K Derham1, J Clive Ellory2, Anthony J Bron1and John J Harding1
1
Nuffield Laboratory of Ophthalmology, University of Oxford, UK;2Laboratory 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-crysa-crys-tallin was
incorporated into red cell ghosts 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-sensitive86Rb 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/Kpump Intracellular a-crystallin protected against the decrease in ouabain sensi-tive86Rb 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 86Rb (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 lM) methylglyoxal binds to proteins in blood plasma [6]
Reactive oxygen species such as hydrogen peroxide (H2O2) 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 aA and aB 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: john.harding@eye.ox.ac.uk
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)
Trang 2between 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, H2O2
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
86Rb 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 4C 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 0C with isotonic Hepes buffer (20 mMHepes,
146 mMNaCl, 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 mMPipes, 0.1 mMEDTA 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 0C by a cooling jacket with
circulating antifreeze The column was eluted with Hepes buffer at a constant flow rate of 30 mLÆh)1and 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, 10 min, 0C), the supernatant aspirated and the pellet re-suspended in isotonic Hepes buffer at 0C to 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 0C Resealing buffer contained NaCl (10 mM), KCl (140 mM), Mops (10 mM), dithiothreitol (2 mM), EGTA (0.1 mM), potassium phosphate (1 mM) and MgCl2 (0.15 mM) at pH 7.4 Potassium ATP (2 mM), sodium phosphocreatine (5 mM) 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) were used instead
of a-crystallin
The tubes containing the suspensions were placed on ice for 10 min then at 37C for 30 min so that the lysed ghost cells would reseal After resealing the ghost cells were washed three times in Mops buffer (10 mMMops, 146 mM 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.
Trang 3Measurement of86Rb flux while modifying Na/K-ATPase
The activity of Na/K-ATPase was assessed by
ouabain-sensitive86Rb uptake Ghost cells were incubated at 37C
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 mM);
(c) modifier; and (d) modifier and ouabain The modifiers
included sucrose (50 mM), fructose (50 mM), methylglyoxal
(0.1 mM, 1 mM and 10 mM) and H2O2 (0.5 mM) All
experiments were performed in triplicate
The Na/K-ATPase flux was initiated by the addition
86Rb and sufficient potassium nitrate to yield a final
concentration of 7.5 mM(20 lCi86Rb per ml buffer) The
tubes were shaken and then incubated at 37C 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 of86Rb by four successive
re-suspensions and centrifugation in ice-cold wash solution
(0.1M MgCl2, 10 mM 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 mMTris/HCl pH 6.8,
5 mM MgSO4, 0.5 mM EDTA, 0.5 mM 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
mMÆL)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, 14C 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 mMMops, 146 mMsodium nitrate) and centri-fuged (10 000 g, 5 min) The ghost cells were then incubated for 2 h at 37C 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 mM) 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-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)1onto the SDS/PAGE to highlight the fact that it can be resealed, 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, 14C sucrose was resealed into ghost cells The cells were washed three times
to remove radioactivity in the supernatant and incubated for 2 h at 37C After this time no radioactivity was detectable in the supernatant At the same time, significant radioactivity of14C sucrose in the ghost cells was measured which was the same at t¼ 0 and 2 h This indicates that the
14C 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 37C, after which time the activity of Na/K-ATPase was measured over a 30-min period by86Rb uptake A steady-state of Na,
86Rb 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]
Trang 4The effects of ouabain, a-crystallin and sugars on the
86Rb uptake of the ghost cells are shown in Fig 3 Ouabain
(0.1 mM), a specific inhibitor of Na/K-ATPase, caused
a 35% decrease in the86Rb 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 in86Rb uptake The presence of
50 mMsucrose, a nonreducing sugar, on86Rb uptake after a
6 h incubation caused no significant decrease in 86Rb
uptake
When the ghost cells were incubated with 50 mMfructose
for 6 h the86Rb uptake was inhibited by 45%, which is
10% greater than that induced by ouabain When the
experiment was repeated with ouabain and 50 mMfructose,
the86Rb 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 mMfructose for 6 h with a-crystallin (1 mgÆmL)1)
resealed inside the ghost cells, the 86Rb 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 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 86Rb uptake was similar to that with fructose alone, i.e 45% inhibition (Fig 3)
When the ghost cells were incubated with 0.1 mM methylglyoxal for 6 h the 86Rb 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 the86Rb uptake into red cell ghosts after 6 h incubation at 37 C, and the effect of the molecular chaperone a-crystallin (1 mgÆmL-1) and BSA ( 1 mgÆmL -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 the86Rb 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.
Trang 5with the addition of ouabain, the86Rb uptake was inhibited
by 35%, indicating that 0.1 mM methylglyoxal did not
inhibit ouabain-sensitive86Rb uptake completely When the
ghost cells were incubated with 0.1 mMmethylglyoxal for
6 h with a-crystallin (1 mgÆmL)1) resealed inside the ghost
cells, the86Rb uptake was maintained at 100% of control
(P < 0.05 compared to inhibition by methylglyoxal)
Increasing concentrations of methylglyoxal caused a
dose-dependent decrease in86Rb uptake Incubation with 1 mM
methylglyoxal inhibited86Rb 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 mM
methylglyoxal86Rb uptake was restored to 90% of the
control (P < 0.05 compared to inhibition by
methylgly-oxal) At 10 mMmethylglyoxal the86Rb uptake of the ghost
cells was inhibited by 50%, and the presence of ouabain
did not inhibit it further This suggests that 10 mM
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
inside86Rb 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 H2O2 When the ghost cells were incubated with 0.5 mM
H2O2for 6 h the86Rb 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 mM H2O2 for 6 h with a-crystallin
(1 mgÆmL)1) resealed inside the ghost cells, the86Rb uptake
was restored to 85% of control (P < 0.01 compared to
inhibition by H2O2) When the ghost cells were incubated
with 0.5 mM H2O2, ouabain and a-crystallin, the 86Rb
uptake was approximately the same, as that with ouabain
alone, indicating a selective effect of H2O2on the Na, K pump
To ensure that changes in 86Rb 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 86Rb 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 86Rb 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 lL samples taken
at t ¼ 0 and t ¼ 6 h and assayed Error bars represent standard deviation.
Trang 6Flame 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 mM, which did not decrease over
6 h All modifiers had zero time Na+ concentrations of
100 mM that did not decrease over 6 h The K+
concentration in all ghost cells, control and modified, were
13 mMat time zero and 9 mMat time 6 h Thus the
changes in86Rb 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 H2O2 and activity measured by86Rb
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 H2O2 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 in86Rb flux caused by fructose,
methyl-glyoxal and HO 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 86Rb in the ghost cells with ouabain (0.1 mM) decreased by 35% This is the amount of86Rb flux across the ghost cell membrane contributed by Na/K-ATPase The other 65% of Kinflux presumably reflects an increased Kleak 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 H2O2 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] H2O2oxidizes 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 toD-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
Trang 7synthesis is not inhibited [32] Heat shock of Reuber H35
hepatoma cells did not cause decrease in ouabain-sensitive
86Rb 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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