Saturation transfer EPR studies of membrane alteration in hereditary spherocytosis

Our earlier spin label electron paramagnetic resonance (EPR) studies of hereditary spherocytosis (HS) erythrocyte revealed the existence of structural alteration(s) when the membrane is subjected to heat stress. We have now used saturation transfer EPR to show restricted motion in membrane proteins even without subjecting membrane to stress. The abnormal motional behavior is amplified when membranes are incubated at 47 degrees C and is readily detectable by conventional EPR. Gel electrophoresis and lipid assays show that proteins but not lipids are released upon heating. Thus, the more restricted motions in HS membranes may be due to a different membrane protein organization, ultimately resulting in the abnormal morphology of HS cells.     

Studies on protein kinase activity and the binding of adenosine 3'5-monophosphate by membranes of hereditary spherocytosis erythrocytes.

Evidence has been presented recently of a deficiency of an endogenous membrane-associated protein kinase in erythrocytes of patients with hereditary spherocytosis (HS). We have measured endogenous protein kinase activity in erythrocyte membranes of 4 HS subjects using different membrane isolation and reaction conditions and find that the phosphorylation of the spectrin component (mean ± S.E. 17.1 ± 1.2 pmoles/10 mins per mg protein) is not significantly different to that of 4 normal controls (mean ± S.E. 20.7 ± 1.1 pmoles/10 mins per mg protein). Phosphorylation of exogenous proteins such as casein and protamine is also not deficient in HS erythrocyte membranes. Adenosine 3′5-monophosphate (cyclic AMP) binding to normal and HS erythrocyte membranes was also studied using a Millipore filtration assay. The affinity of cyclic AMP for erythrocyte membranes as determined by Hill plots of binding data from 4 HS subjects (K_D mean ± S.E. = 2.2 ± 0.2 nM) was not significantly different to 4 normal controls (K_D mean ± S.E. = 2.8 ± 0.6 nM). The rate of dissociation of bound cyclic AMP from HS membranes was also similar to control membranes. We thus cannot confirm the prediction by others that an abnormality of cyclic AMP interaction with the erythrocyte membrane underlies HS.     

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 H(2)O(2); 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.     

Irradiation increases proteolysis in erythrocyte ghosts: A spin label study

Summary X- and gamma-irradiation of human erythrocyte membranes (250–1000 Gy) was found to decrease the ratio of weakly to strongly immobilized signal height of membrane-bound maleimide spin label (Mal-6). Subsequent incubation of spin-labeled membranes at ambient temperature (21 °C) induced a progressive increase in this ratio, faster for membranes irradiated with low doses which was hampered by protease inhibitors. These results demonstrate that ionizing radiation stimulates proteolysis of erythrocyte membrane proteins by membrane-associated proteases.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

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.     

Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects

Mobilization and aggregation of intramembrane particles (IMPs) are physiological events observed in various cells. In erythrocyte membranes, aggregation of IMPs can be induced by the exposure of partially desprectrinized erythrocyte membranes to acidic pH. We investigated the association between IMPs aggregation, protein mobility, and membrane fluidity in erythrocyte membranes of healthy controls and Duchenne muscular dystrophy (DMD) patients by using electron spin resonance and specific spin labels for membrane proteins and lipids. In erythrocyte membranes of control subjects, the partial spectrin removal induced a decreased segmental motion of protein spin label indicating an increase of protein-protein interactions. Stearic acid spin labels 5- and 16-(N-oxyl-4,4'-dimethyloxazolidine) showed that the treatment induces an increase of membrane fluidity. In DMD patients, both treated and untreated erythrocyte membranes showed changes of membrane fluidity when compared to those of the controls. Our results suggest that defects in the interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD.     

Erythrocyte membrane alterations in Huntington disease: Effects of γ-aminobutyric acid

The interaction of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) with erythrocyte membranes from patients with Huntington disease and normal controls has been studied by electron spin resonance. GABA affects the physical state of erythrocyte membrane proteins in control and Huntington disease differently. In addition, after exposure of spin-labeled Huntington disease erythrocyte membranes to 0.1 mM GABA, the relevant electron spin resonance parameters reflecting the physical state of membrane proteins are indistinguishable from those of untreated control membranes. These findings support the concept that this disease is associated with a generalized membrane defect.     

Changes in erythrocyte membranes after fractioned hyperthermia

The structural changes in erythrocytes membranes were examined before and after the second heat shock of erythrocytes. Electrophoretic separation of protein erythrocyte membranes for cells incubated at 48.5°C was different from control i.e. from erythrocytes incubated at 37°C. No quantitative or qualitative changes were spotted in comparison with protein membranes isolated from the erythrocytes following single or double heat shock. Fluidity of erythrocytes membranes was determined by using spin labels, 5-doxylstearic acid and 16-doxylstearic acid. The membranes were more rigid in their hydrophobic regions after incubation of cells at 44°C. It can be suggested that erythrocyte membranes play some role in thermotolerance and heat damage of enuclate cells.     

Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane

The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant.     

Time course of the age-related alterations in stored blood

The extent and time course of the impairments occurring in whole blood and erythrocyte cells stored under blood bank conditions were studied by monitoring the reduction of MAL-6 spin label added to the media containing whole blood or erythrocyte cells using electron spin resonance (ESR) technique. Impairments forming in the erythrocyte cells incubated for various times at 37 degrees C were also studied. Erythrocyte cells were found to undergo changes during the storage or incubation, leading to fast decay of MAL-6 spin labels signal height. The extent of the changes depends on storage or incubation time. However, the reduction in incubated or artificially aged erythrocyte (AAE) cells was faster than the reduction in whole blood (WB) and aged erythrocyte (AE) cells stored under blood bank conditions. Two exponential curves attributed to the liquid and cellular parts of a given samples were found to be described best in the reduction of MAL-6 spin label in WB, AE and AAE.     

The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.     

Erythrocyte membrane in Duchenne muscular dystrophy. II. Fluidity of lipids in dystrophic patients and their families

Electron spin resonance techniques was used to study the fluidity of intact and hemoglobin free erythrocyte membranes from patients with Duchenne muscular distrophy and from members of their family. A greater mobility of spin label motion was observed only at the surface of hemoglobin free membranes in the patients. These studies suggest alterations in lipid-protein interaction, indicating in Duchenne muscular distrophy a generalized membrane abnormality.     

Low-pH association of proteins with the membranes of intact red blood cells. I. Exogenous glycophorin and the CD4 molecule

Glycophorin and CD4 proteins are tightly associated with intact human erythrocyte membranes after a short-time incubation at low pH (1-2 min, pH lower than 5, 37 degrees C). Flow cytometry and epifluorescence microscope observations showed that after incubation of red cells with fluorescein isothiocyanate (FITC) labeled glycophorin at pH values lower than 5, the erythrocyte membrane and subsequently formed ghost membranes were fluorescent. Unlabeled glycophorin was reacted with mouse erythrocytes using the same low-pH conditions. Flow cytometry and fluorescence microscopy showed that anti-glycophorin monoclonal antibodies were able to recognize the epitopes of glycophorin associated with the mouse erythrocytes. Kinetic experiments showed that the interaction of FITC-glycophorin with red cell membranes can be monitored by a decrease in the fluorescence intensity. Erythrocyte associated glycophorin was not removed from the membranes after 24 h incubation in human plasma (in vitro, 39 degrees C). A glycoprotein extract containing CD4 was isolated from a T4-lymphoma cell line (CEM). This protein extract was incubated with erythrocytes using the same low-pH conditions. Fluorescently labeled monoclonal antibodies against CD4 stained the red cells after association of CD4 with the membranes. Electron microscopy showed 10 nm immunoglobulin G-coated gold beads associated with CD4-bearing erythrocyte membranes after incubation with anti-CD4 antibodies and then with the gold beads. The potential use of the CD4-erythrocyte complex as a therapeutical agent against acquired immune deficiency syndrome (AIDS) is suggested.     

Effect of fatty acyl chain length of phosphatidylcholine on their transfer from liposomes to erythrocytes and transverse diffusion in the membranes inferred by TEMPO-phosphatidylcholine spin probes

TEMPO-phosphatidylcholine (PC) spin probes which have homologous saturated acyl chains of 10, 12, 14 and 16 carbon atoms, were synthesized as analogues of PC. Transfer of TEMPO-PCs from liposomal membrane to the ghost membrane of human erythrocyte and transverse diffusion of TEMPO-PCs within the membrane of intact erythrocytes were determined by measurement of spontaneous increase and decrease in signal amplitude of an anisotropic triplet spectrum, due to dilution of the label by natural phospholipid of the membrane and reduction of the label by the cytoplasmic content of the erythrocyte, respectively. TEMPO-PC molecules in TEMPO-PC liposomes, except dipalmitoyl TEMPO-PC, were rapidly incorporated into the ghost membrane by incubation at 37 degrees C; the PC having shorter acyl chains was transferred faster. The cytoplasmic content of the erythrocyte rapidly reduced the nitroxide radical of the spin probe. The central peak height of ESR signal was once increased by incorporation of TEMPO-PC into the erythrocyte membrane and then was spontaneously decreased during further incubation at 37 degrees C. This decrease indicates that PC molecules traverse from the outer to the inner layer of the membrane lipid bilayer. The decrease of signal amplitude was faster with PC of shorter acyl chain. These findings suggest that both transfer between membranes and transverse diffusion in the membrane may be favored to the PC species with shorter acyl chains.     

Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.     

Carriers of ataxia-telangiectasia gene display additional protein fraction and changes in the environment of SH groups in erythrocyte membrane

Additional protein fraction migrating slower than spectrin has been detected in erythrocyte membranes from an ataxia-telangiectasia (A-T) patient and from his mother (A-T heterozygote). In erythrocyte membranes labelled with maleimide spin label changes in signal of the weakly immobilized spin label as related to that of strongly immobilized one (w/s) were noted. In comparison to age-matched control groups the values of w/s were lower in A-T heterozygotes (ten persons) and higher in A-T homozygotes (four persons). In control persons the values of w/s increased with age, whereas in families with A-T no significant differences in this parameter were noted between children and parents. The presence of additional protein fraction in erythrocytes membranes of A-T patient and A-T heterozygote indicates that these phenotypes can be differentiated from the healthy control persons for the first time on the basis of changes detected in the erythrocytes. This change in erythrocyte membrane may explain the decrease in the w/s parameter of electron spin resonance in A-T heterozygotes. On the other hand increased values of w/s in A-T patients may be caused by disease process.     

The mobility and reactivity of maleimide-binding proteins in the rat erythrocyte membrane. Effects of dietary zinc deficiency and incubation with zinc in vitro

Erythrocyte ghosts, prepared from rats fed zinc-deficient diets, were analyzed for the mobility of membrane proteins by electron spin resonance spectroscopy of the sulfhydryl-binding spin probe, 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Compared with erythrocyte membranes from rats fed zinc-adequate diets ad libitum or pair-fed, erythrocyte membranes from zinc-deficient rats had a significantly increased ratio of weakly immobilized to strongly immobilized probe-binding proteins. This suggests that dietary zinc deficiency causes a conformational change in erythrocyte membrane proteins. Dietary zinc deficiency did not significantly affect N-ethylmaleimide (NEM)-induced thermal sensitivity or NEM-induced mechanical fragility in rat erythrocytes; however, the addition of zinc in vitro to red cells significantly inhibits NEM-induced mechanical fragility.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

The effects of 3-methylindole on hemolysis, transport of Na+, and ATPase activities of bovine erythrocytes.

Biochemical effects of 3MI on cellular membranes were investigated. This study was conducted to examine the effects of 3MI on the hemolysis of erythrocytes, the transport of 22Na+ in resealed erythrocyte ghosts, and on the ATPase activities of erythrocyte membranes. The percent of hemolysis as a function of 3MI incubation time was sigmoidal. Seventy-five percent of the hemoglobin was released with the second 2 hr of incubation during which the concentration of 3MI in the cells reached a plateau of 2500 mug/ml of packed RBC. The effect of 3MI at a subhemolytic concentration on passive and active 22Na+ transport were not significant. The total and Mg2+-dependent ATPase activities in the membranes were significantly increased after 1 hr of incubation with 3MI at concentrations of 100, 200, 300, 400 and 500 mug/ml (P less than or equal to 0/ml (P smaller than or equal to 0.02).     

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.