Isolated Erythrocyte Membranes Research Articles

IMPACT OF HYPERGLYCEMIA ON ERYTHROCYTE'S MEMBRANE BAND 3 PROTEIN (BP3)

To prevent T2DM and its chronic complications early diagnostic of carbohydrates metabolism disorders is necessary. We investigated the erythrocytes membrane proteins in groups of patients with insulin resistance, prediabetes, and T2DM. Blood was obtained from healthy volunteers and patients with T2DM, prediabetes, and IR. Band 3 protein (B3p) content in isolated erythrocyte membranes was determined with the analytical electrophoresis method. АNOVA was used for the comparative analysis of the data. In patients with disorders of carbohydrate metabolism the expression of B3p (90-100kDa) was reduced in comparison to the control level and appeared as the oligomerized form of B3p (180kDa). Therefore, it seems that in the process of modification of erythrocyte membranes of patients with disorders of carbohydrate metabolism participate both, hyperglycemia and oxidative stress conditions associated with diabetes.

СТРУКТУРНО-ФУНКЦИОНАЛЬНОЕ СОСТОЯНИЕ БЕЛКОВ В ЭРИТРОЦИТАРНЫХ МЕМБРАНАХ ПАЦИЕНТОВ С АРТЕРИАЛЬНОЙ ГИПЕРТЕНЗИЕЙ

Objective. To study the structural and functional state of proteins in erythrocyte membranes of AH patients with asymptomatic brain lesions (ABL) and AH patients without brain lesions who suffered from cerebrovascular accident (CVA). Methods. The study included 47 patients diagnosed with AH. The study groups were formed based on the detected neuroimaging changes in the brain and analysis of the structural and functional state of proteins in the membranes of erythrocytes in patients with AH. The following groups were identified: 1-рatients with AH without BL 2-patients with AH and ABL 3-patients with previous cerebrovascular accident Results. A comparative analysis of abnormalities of structural and functional state of proteins in erythrocyte membranes in patients with AH without brain lesions, in patients with AH with asymptomatic brain lesions visualized by MRI, and patients who suffered from cerebrovascular accident showed the following: 1. In patients with AH with asymptomatic brain lesions, and in those suffered from cerebrovascular accident, the value of the maximum reaction rate (Vmax) of AChE in erythrocyte membranes was significantly reduced in comparison with the values of this parameter in patients with AH without brain lesions (Figure 1). 2. The value of Michaelis constant (MC) for AChE in isolated erythrocyte membranes of patients with AH without brain lesions and with asymptomatic brain lesions is increased compared with the group of patients with a history of cerebrovascular accident (Figure 2). Conclusion. In patients with hypertension, the structural and functional state of proteins in erythrocyte membranes is disturbed, depending on the severity of the damage to the brain.

Erythrocyte membranes convert monomeric ferriprotoporphyrin IX to β-hematin in acidic environment at malarial fever temperature

Hemozoin production makes it possible for intraerythrocytic malaria parasites to digest massive quantities of hemoglobin but still avoid potential ferriprotoporphyrin IX (FP) toxicity, which they cannot decompose further. Some antimalarial drugs, such as chloroquine, work by inhibiting this production, forcing the parasite to starve to death. As part of the efforts to identify possible biological mechanisms of FP polymerization, we have used normal human erythrocyte membranes as a model, to promote β-hematin (β-h) synthesis. Hemin in 35% aqueous dimethyl sulfoxide (DMSO) was reacted with isolated erythrocyte membranes and incubated overnight in sodium acetate buffer, pH 4.8, at 41°C. Infrared spectroscopy and electron microscopy showed that β-h was produced. Hemin in 10% was less effective as the substrate than when it was in 35% DMSO. A high malarial temperature seemed to be necessary, because FP polymerization was less at 37°C than at 41°C. Production was partially inhibited by chloroquine. These observations are of interest because other investigators have reported that membrane lipids mediated FP polymerization, but whole membranes were ineffective. On the other hand, our hypothesis is that the transport vesicles (TV) in malaria parasites could provide the receptor for FP and the lipids that promote hemozoin formation. Erythrocyte membranes may not be directly involved, but Plasmodium species transport hemoglobin in membrane-bound TV into food vacuoles, where hemoglobin catabolism is completed and hemozoin crystals are stored.

End products of lipid peroxidation in erythrocyte membranes in Alzheimer's disease

Alzheimer's disease (AD) is accompanied by oxidative stress in the brain. Because the brain tissue is rich in polyunsaturated fatty acids, it is prone to the free radical attack resulting in lipid peroxidation. Intermediates of lipid peroxidation may diffuse from the primary site, cross the blood-brain barrier and modify erythrocyte membranes in the bloodstream. We exposed isolated erythrocyte membranes from patients with AD and the control group to in vitro free radical damage and monitored the accumulation of the end products of lipid peroxidation, lipofuscin-like pigments (LFPs), by fluorescence spectroscopy. LFPs were analyzed by means of tridimensional and synchronous fluorescence spectroscopy. The levels of LFP formed during in vitro peroxidation were significantly higher in erythrocyte membranes from patients with AD compared with the control group. Furthermore, the chemical composition of LFP in AD was different from the control group. The analysis of the specific modifications of erythrocyte membranes in AD is of great medical importance regarding the need of a diagnostic blood biomarker.

Plasma lipid pattern and red cell membrane structure in β-thalassemia patients in Jakarta

Background: Over the last 10 years, we have investigated thalassemia patients in Jakarta to obtain a comprehensive picture of iron overload, oxidative stress, and cell damage. Methods: In blood samples from 15 transfusion-dependent patients (group T), 5 non-transfused patients (group N) and 10 controls (group C), plasma lipids and lipoproteins, lipid-soluble vitamin E, malondialdehyde (MDA) and thiol status were measured.  Isolated  eryhtrocyte  membranes  were  investigated  with  electron  paramagnetic  resonance  (EPR)  spectroscopy  using doxyl-stearic acid and maleimido-proxyl spin lables. Data were analyzed statistically with ANOVA. Results: Plasma triglycerides were higher and cholesterol levels were lower in thalassemic patients compared to controls. Vitamin  E,  group  C:  21.8  vs  T:  6.2  μmol/L)  and  reactive  thiols  (C:  144  vs.  T:  61  μmol/L)  were  considerably  lower  in  transfused patients, who exert clear signs of oxidative stress (MDA, C: 1.96 vs T: 9.2 μmol/L) and of tissue cell damage, i.e.,  high  transaminases  plasma  levels.  Non-transfused  thalassemia  patients  have  slight  signs  of  oxidative  stress,  but  no  signifi  cant  indication  of  cell  damage.  Erythrocyte  membrane  parameters  from  EPR  spectroscopy  differ  considerably  between all groups. In transfusion-dependent patients the structure of the erythrocyte membrane and the gradients of polarity and fl uidity are destroyed in lipid domains; binding capacity of protein thiols in the membrane is lower and immobilized. Conclusion: In  tranfusion-dependent  thalassemic  patients,  plasma  lipid  pattern  and  oxidative  stress  are  associated  with structural damage of isolated erythrocyte membranes as measured by EPR spectroscopy with lipid and protein-thiol spin labels. (Med J Indones 2011; 20:178-84)

The Proteasome Is an Integral Part of Solar Ultraviolet A Radiation-induced Gene Expression

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

ATPase and acetylcholinesterase activities in erythrocyte membranes after incubation with glucose and in streptozotocin diabetic rats.

Enzyme activities of ATPases and acetylcholinesterase from isolated erythrocyte membranes (ghosts) were investigated before and after incubation with 50 mM glucose. Glucose incubation caused a time dependent loss of ATPase and acetylcholinesterase activities. Ghost enzyme activities in steptozotocin diabetic rats were found only insignificantly diminished.

Hypercapnia protects erythrocytes against free radical damage induced by hypoxia in exposed rats

Several studies report that hypoxic exposure induces free radical oxidative damage in various tissues. The mechanism of this damage includes membrane lipid peroxidation which can be easily detected by measuring fluorescent end-products of the process, i.e. lipofuscin-like pigments. Four day exposure of rats to hypoxia (10% O(2)) increased the level of lipofuscin-like pigments in erythrocytes up to 9 fold. This increase was completely prevented when the animals were exposed to hypercapnia (4.3% CO(2)) in addition to hypoxia. We studied the possible mechanism of the hypercapnic protection on isolated erythrocyte membranes in vitro. Lipid peroxidation was initiated by incubation of the membranes with iron ions and ascorbate. Production of malonaldehyde, the precursor of lipofuscin-like pigments, was strongly inhibited in bicarbonate buffer. Similarly the production of lipofuscin-like products was damped. These experiments suggest that the protective effect of hypercapnia might consist in direct interaction of CO(2) with free radical processes.

The interaction of DNR and glutaraldehyde with cell membrane proteins leads to morphological changes in erythrocytes

In this study, the effects of DNR and glutaraldehyde on isolated erythrocyte membrane proteins were examined. For this purpose, SDS–gel electrophoresis was carried out. Additionally, analyses of the disturbances in erythrocyte shape and size, accompanied by the application of flow cytometry and microscopy examination, were undertaken. The amount of DNR linked to erythrocyte cell membranes was measured by a fluorimetric technique. It was observed that glutaraldehyde caused in concentration dependent manner an increase of percent of DNR linked to cell membrane proteins. After this incorporation, perturbations in the protein content of cell membranes were observed. The protein aggregates and changes in the level of spectrin, actin and band 3 protein were noted. Due to the changes in spectrin, which is mainly responsible for maintenance of the discocyte shape of erythrocytes, flow cytometry and microscopy techniques were used to control the size and shape of erythrocytes after treatment with DNR and glutaraldehyde. The disturbances in the shape and size of erythrocytes were observed for all tested concentrations of glutaraldehyde. For all tested concentrations of glutaraldehyde, the changes were statistically significant.

The oxidative damage of butenolide to isolated erythrocyte membranes

Butenolide (CAS No. 16275-44-8), a mycotoxin produced by several Fusarium species, has been shown to be a potential risk factor for animal and human health. This study was undertaken to investigate the potential oxidative damage of butenolide to biomembranes in vitro using the erythrocyte membrane model. Following exposure of isolated rat erythrocyte membranes to butenolide, the extent of oxidative damage was assessed by measuring lipid peroxidation, –SH groups content, Ca 2+/Mg 2+-ATPase and Na +/K +-ATPase activities, and conformational changes in membrane proteins. It was observed that butenolide resulted in a significant lipid peroxidation, revealed by a concentration-dependent increase in the level of thiobarbituric acid reactive substances (TBARS). Similarly, this toxin induced a concentration-dependent decrease in the content of membrane total –SH groups, as well as free –SH groups. Membrane-bound enzymes were also impaired by the toxin, demonstrated by the marked inhibition of the activities of Na +/K +-ATPase and Ca 2+/Mg 2+-ATPase. Conformational changes in membrane proteins were determined using electron paramagnetic resonance (EPR) spin labeling. Butenolide caused an increase in the ratio of weakly to strongly immobilized components ( W/ S ratio) in a manner of concentration-dependent, indicating conformational changes in membrane proteins occurred. In conclusion, these findings indicate that butenolide is capable of inducing significant oxidative damage to membrane lipids and proteins.

Photoinduced modifications by fluoroquinolone drugs in bovine serum albumin (BSA) and ribonuclease A (RNAse) as model proteins

Photosensitized protein oxidation by drugs, with the consequent modification of their structure is thought to be responsible for the occurrence of phototoxic phenomena such as photoallergy and loss of biological functions. In this paper we have investigated in detail the interaction of four fluoroquinolones namely ciprofloxacin, lomefloxacin, norfloxacin and ofloxacin with two proteins such as Bovine Serum Albumin (BSA) and Ribonuclease A (RNAse A) chosen as models. The interactions between the four drugs and the proteins, were studied by absorption and emission spectroscopy. Photophysical experiments were carried out in aqueous solutions by stationary and time-resolved fluorimetry and by laser flash photolysis, in the absence and in the presence of the proteins to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed upon irradiation. In parallel we have investigated by a series of biochemical assays the photoinduced modifications exerted by the four drugs. The obtained results showed that the four drugs are able to photooxidize proteins with the formation of protein-protein cross-link. This effect was also confirmed in isolated erythrocyte membranes. Furthermore the effect of the fluoroquinolones was also evaluated on isolated aromatic aminoacids. In this context the four drugs are able to photodamage in particular tyrosine and histidine. These results are important in the light of the growing interest for the comprehension of the mechanism of phototoxicity induced by these antibacterial drugs. * Prof. Francesco Dall’Acqua Department of Pharmaceutical Sciences, University of Padova Via Marzolo 5, 35131-Padova ITALY Tel +39 49 8275708, Fax: +39 49 8275366 E-Mail: Francesco.dallacqua@unipd.it Issue in Honor of Prof. Waldemar Adam ARKIVOC 2007 (viii) 231-244 ISSN 1424-6376 Page 232 ARKAT USA, Inc.

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H2O2; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.

Modulation of human erythrocyte Ca2+-ATPase activity by glycerol: The role of calmodulin

The effect of an intracellular cryoprotectant glycerol on human erythrocyte Ca2+-ATPase activity and possible involvement of calmodulin in the regulation of Ca2+-pump under these conditions were investigated. The experiments were carried out using saponin-permeabilized cells and isolated erythrocyte membrane fractions (white ghosts). Addition of rather low concentrations of glycerol to the medium increased Ca2+-ATPase activity in the saponin-permeabilized cells; the maximal effect was observed at 10% glycerol. Subsequent increase in glycerol concentrations above 20% was accompanied by inhibition of Ca2+-ATPase activity. Lack of stimulating effect of glycerol on white ghost Ca2+-ATPase may be attributed to removal of endogenous compounds regulating activity of this ion transport system. Inhibitory analysis using R24571 revealed that activation of Ca2+-ATPase by 10% glycerol was observed only in the case of inhibitor administration after modification of cells with glycerol; in the case of inhibitor addition before erythrocyte contact with glycerol, this phenomenon disappeared. These data suggest the possibility of regulation of human erythrocyte Ca2+-ATPase by glycerol; this regulatory effect may be attributed to both glycerol-induced structural changes in the membrane and also involvement of calmodulin in modulation of catalytic activity of the Ca2+-pump.

The response of Na+/K+-ATPase of human erythrocytes to green laser light treatment

The objective of this study was to investigate the response of Na(+)/K(+)-ATPase of human erythrocytes to green laser irradiation. Effects of green laser light of fluences 9.5-63.3 J.cm(-2) and merocyanine 540-mediated laser light treatment were studied. Isolated erythrocyte membranes (protein concentration of 1 mg/ml) were irradiated by Nd:YAG laser (532 nm, 30 mW) and then incubated in a medium with 2 mM ATP for 30 min. Activity of ATPase was determined colorimetrically by measuring the colored reaction product of liberated inorganic phosphate and malachite green at 640 nm. Contribution of Na(+)/K(+)-ATPase to overall phosphate production was determined using ouabain. A positive effect of green laser light on Na(+)/K(+)-ATPase activity was observed. The dependence of enzymatically liberated inorganic phosphate on light fluence showed a linear correlation (R(2)=0.96, P=0.0005) for all fluences applied (9.5-63.3 J.cm(-2)). On the other hand, MC 540-mediated phototreatment caused a suppression of enzyme activity.

Membrane Oxidation Assay--A Novel Lipoxygenase-based Evaluation of Membrane Oxidizability

A membrane oxidation assay is presented which uses isolated erythrocyte membranes ("ghosts") and lipoxygenase as a selective catalyst for the transfer of oxygen to cis - cis -1,4-pentadiene-moieties. The latter are, for instance, present in linoleic and arachidonic acids, both of which are integral parts of membranes. These non-conjugated double bonds represent energetically favorable sites for oxidative attack and therefore, may be rearranged and partially consumed during oxidative stress. Consequently, the measurement of oxygen consumption in the course of the lipoxygenase-mediated oxidation provides a tool for the quick and reliable determination of such double bonds. Significant inter-individual differences have been noted in 11 subjects, which also correlate to the total radical antioxidant parameter (TRAP) values obtained. This assay will be helpful in the assessment of oxidizable structures in erythrocyte membranes that may be diminished as a consequence of oxidative damage suffered by an individual. In conclusion, a simple and rapid assay for the assessment of the oxidizability of erythrocyte membranes is presented complementing the TRAP assay for plasma antioxidative status.

Isolated erythrocyte membranes of transfusion-dependent and non-transfused thalassemia patients in Jakarta, investigated by electron paramagnetic resonance spectroscopy.

Erythrocyte membrane structural parameters were studied in transfusion-dependent beta-thalassemia patients, in long-term transfused patients (regularly transfused < 15 years), and in those who had not yet obtained transfusions. Controls were voluntary students up to 30 years of age without diagnosis or clinical signs of thalassemia. Membranes were isolated and investigated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron paramagnetic resonance (EPR) spectroscopy. Data obtained from the thiol-reactive spin label N-ethyl-maleimidoproxyl reveal immobilization of protein environment in erythrocyte membranes from thalassemic patients. SDS-PAGE shows both degradation and aggregation of membrane proteins. Thalassemic erythrocyte membranes exert higher order parameters in the hydrophobic region as determined by 16-doxyl-stearic acid. Rotational correlation times of this spin label increase only in transfused patients. Polarity is higher in membranes of all patients than in controls. In the polar interface, order parameters obtained from 5-doxyl-stearic acid increase in non-transfused and decrease in transfusion-dependent patients as compared with controls. Transfused patients exert increasing membrane order in the hydrophobic region and counter-currently decreasing order in the polar interface indicating loss of membrane integrity along with the loss of fluidity and polarity gradients and the loss the energetic barrier function of the membrane.

Erythrocytes as targets for gamma-glutamyltranspeptidase initiated pro-oxidant reaction.

Gamma-glutamyltranspeptidase (GGT) is a well known cell plasma membrane and serum circulating enzyme. In clinical chemistry, GGT is used as a marker of alcohol consumption and drug uptake. Serum GGT activity varies in hepatobiliary diseases and cancer. This enzyme is involved in glutathione (GSH) metabolism, which is generally associated with antioxidant properties. However, in recent years, findings from our group and from others showed that GGT-catalysed extracellular metabolism of GSH leads, in the presence of iron, to the generation of reactive oxygen species (ROS). It was demonstrated that those highly reactive species oxidise lipids, cell surface protein thiols or activate transcriptional factors such as Nuclear Factor kappaB (NFkappaB). The objective of the present work is to determine whether the red blood cells are targets for plasma GGT-initiated pro-oxidant reaction. The results obtained demonstrate that the GGT/GSH/iron system oxidises isolated erythrocyte membranes. A significant release of haemoglobin and a decrease of erythrocyte deformability are also observed. In addition, in vivo studies showed a relationship between plasma GGT activity and erythrocyte deformability in 20 studied subjects. In conclusion, GGT-mediated ROS production is able to oxidise erythrocytes and thus disturbs their functions.

Antennapedia/HS1 chimeric phosphotyrosyl peptide: conformational properties, binding capability to c-Fgr SH2 domain and cell permeability.

With the aim of interfering with the signaling pathways mediated by the SH2 domains of Src-like tyrosine kinases, we synthesized a tyrosyl-phospho decapeptide, corresponding to the sequence 392-401 of HS1 protein, which inhibits the secondary phosphorylation of HS1 protein catalyzed by the Src-like kinases c-Fgr or Lyn. This phospho-peptide was modified to enter cells by coupling to the third helix of Antennapedia homeodomain, which is able to translocate across cell membranes. Here we present CD and fluorescence studies on the conformational behavior in membrane-mimicking environments and on lipid interactions of Antennapedia fragment and its chimeric phosphorylated and unphosphorylated derivatives. These studies evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption on lipids. Experiments performed with recombinant protein containing the SH2 domain of c-Fgr fused with GST and with isolated erythrocyte membranes demonstrated that the presence of the N-terminal Antennapedia fragment only slightly affects the binding of the phospho-HS1 peptide to the SH2 domain. In fact, it has been shown that in isolated erythrocyte membranes, both phospho-HS1 peptide and its chimeric derivative greatly affect either the SH2-mediated recruitment of the c-Fgr to the transmembrane protein band 3 and the following phosphorylation of the protein catalyzed by the Src-like kinase c-Fgr. The ability of the chimeric phospho-peptide to enter cells has been demonstrated by confocal microscopy analysis.

Mechanism of band 3 dimer dissociation during incubation of erythrocyte membranes at 37 °C

The mechanism of dissociation of the stable dimer of band 3 was investigated during the incubation of isolated erythrocyte membranes or resealed ghosts at 37 degrees C. The kinetics of changes in the structural and functional integrity of the membrane domain of band 3 (MDB3) were measured and correlated with the change in the Stokes radius of band 3. MDB3 integrity was determined as follows: (1) by measuring the fluorescence emission spectrum of 4, 4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) bound covalently to MDB3; (2) by measuring the number of DIDS covalent binding sites present after incubation of unlabelled resealed ghosts; and (3) by measuring the anion transport V(max) by using the same resealed ghosts. Incubation of membranes at 37 degrees C caused the dissociation of band 3 dimers to monomers but only after a lag period lasting approx. 50 h. The observation of such a lag implies that dissociation involves a sequence of molecular events beginning with some type of initial process. We have discovered that this initial process involves a conformation change in MDB3. There was a shift in the fluorescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS binding capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 degrees C inhibited the conformational change in MDB3 and the dissociation of dimers. Furthermore, no conformational change in MDB3 was observed when erythrocytes were incubated at 37 degrees C. We suggest that MDB3 unfolding is the molecular event responsible for the subsequent dissociation of stable dimers of band 3 to monomers during the incubation of erythrocyte membranes at 37 degrees C. The monomers so generated are either not functional in anion exchange or they have an attenuated functionality. The absence of a conformational change for band 3 in erythrocytes might imply that haemolysis perturbs the membrane structure and somehow predisposes band 3 to the conformational change that occurs during incubation at 37 degrees C.

Resolution of the paradox of red cell shape changes in low and high pH

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3–7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[ 125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.