ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

Chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

Effects of pH on membrane fluidity of human erythrocytes

The effects of pH on the membrane fluidity of intact human erythrocytes, ghosts, and their lipid vesicles were studied by spin label techniques in the range of pH 3.0 to 9.1. Two fatty acid spin labels, 5-nitroxide stearic acid (5NS) and 12-nitroxide stearic acid (12NS), and a maleimide spin label were used for the labeling of the membrane lipids and proteins, respectively. The outer hyperfine splitting (T parallel) was measured as a parameter of membrane fluidity. In the case of 5NS, the T parallel values for intact erythrocytes and ghosts remained almost constant over the entire pH range at 22 degrees C but those for their lipid vesicles changed slightly, indicating the vertical displacement of the labels in lipid bilayers. On the other hand, the ESR spectra of 12NS incorporated into intact erythrocytes and ghosts, as compared with their lipid vesicles, showed marked pH dependence. By means of spin labeling of membrane proteins, the conformational changes of the proteins were observed in the pH range mentioned above. These results suggest a possible association between the strong pH dependence of the T parallel values and the conformation changes of membrane proteins. The pH dependence of the membrane fluidity was also investigated in cholesterol-enriched and -depleted erythrocytes. The effects of cholesterol demonstrated that the membrane fluidity was significantly mediated by cholesterol at low pH, but not at high pH.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Properties of the strongly immobilized signal observed in spin-labeled erythrocytes

A strongly immobilized signal from fatty acid spin labels was observed in human erythrocytes treated with oxidizing agents such as glutaraldehyde, hydrogen peroxide, phenylhydrazine and copper-ortho-phenanthroline. This signal was also observed in freshly prepared ghosts treated with potassium superoxide and in old erythrocyte ghosts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these samples demonstrated the diffuse, nondiscrete bands of high molecular weight due to the cross-linking of membrane proteins. The temperature and pH dependences of the outer hyperfine splitting of this signal were very similar to those of bovine serum albumin. We propose that the strongly immobilized signal reflects the interaction of the lipids with the cross-linked products of membrane proteins.     

Properties of the strongly immobilized signal observed in spin-labeled erythrocytes

A strongly immobilized signal from fatty acid spin labels was observed in human erythrocytes treated with oxidizing agents such as glutaraldehyde, hydrogen peroxide, phenylhydrazine and copper-ortho-phenanthroline. This signal was also observed in freshly prepared ghosts treated with potassium superoxide and in old erythrocyte ghosts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these samples demonstrated the diffuse, nondiscrete bands of high molecular weight due to the cross-linking of membrane proteins. The temperature and pH dependences of the outer hyperfine splitting of this signal were very similar to those of bovine serum albumin. We propose that the strongly immobilized signal reflects the interaction of the lipids with the cross-linked products of membrane proteins.     

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.     

Electron paramagnetic resonance and saturation transfer electron paramagnetic resonance studies on erythrocytes from goats with and without heritable myotonia

Summary Erythrocytes from myotonic goats, an animal model of heritable myotonia, and normal goats were studied using electron paramagnetic resonance (EPR) and saturation transfer electron paramagnetic resonance (ST-EPR) spin labeling techniques. Three fatty acid spin labels with the nitroxide moiety at progressively greater distances from the carboxyl group were used to monitor different regions within the erythrocyte membrane. Since spin labels have been shown to induce hemolytic and morphologic alterations in erythrocytes, conditions for minimizing these alterations were first defined by hemolysis studies and scanning electron microscopy. Using these defined conditions for our studies we observed no significant differences in any of the EPR or ST-EPR parameters for normal and myotomic goat erythrocytes with any of the fatty acid spin labels used. Our results do not support the theory that myotonia is the result of a generalized membrane defect characterized by increased membrane fluidity as determined by fatty acid spin labels.     

Ionic strength-dependent alterations of membrane structure of red blood cells

Electron paramagnetic resonance (EPR) measurements using various fatty acid spin labels were performed on membranes of intact human erythrocytes at physiological, and at low ionic strength. In the case of spin probes bearing the nitroxide near the polar head group, a less restricted motion at low ionic strength was seen than with those labels with a nitroxide deeper within the hydrophobic tail of the membrane. Although these data clearly show an influence of ionic strength on membrane structure, and possibly a modified protein-lipid interaction, they cannot be simply discussed in terms of an altered membrane fluidity.     

Studies on Tetrahymena membranes:: Temperature-induced alterations in fatty acid composition of various membrane fractions in Tetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study

The fatty acid distribution pattern of lipids extracted from different subcellular components of Tetrahymena pyriformis was found to be significantly different from one type of membrane to another.The growth-temperature shift caused alterations in fatty acid composition. The ratio of palmitoleic to palmitic acid, especially, showed a sharp linear decline with increase of temperature in all of the membrane fractions.The spin labels were rapidly incorporated into Tetrahymena membranes. The order parameter of 5-nitroxide stearate spin label incorporated into various membrane fractions was found to be different for the different membrane fractions, suggesting the following order of the fluidity; microsomes > pellicles > cilia.The fluidity of the surface membranes, cilia and pellicles isolated from Tetrahymena cells grown at 15°C was noticeably higher than that of the membranes from cells grown at 34°C but was not so different with microsomal fractions.The motion of the spin label in the pellicular membrane was more restricted than in its extracted lipids, thus indicating the assumption that in Tetrahymena membranes the proteins influence the fluidity.It was also suggested that a sterol-like triterpenoid compound, tetrahymanol, which is principally localized in the surface membranes, would be involved in the membrane fluidity.     

A study on the reaction of human erythrocytes with hydrogen peroxide

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.     

Motion of spin-labeled fatty acids in murine macrophages. Relation to cellular phagocytic activity.

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels. Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings (2 T11) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives. Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 degrees C, or by fixing them with 0.25% glutaraldehyde. No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with cis- or trans-unsaturated fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

Lipid phase of transverse tubule membranes from skeletal muscle. An electron paramagnetic resonance study.

The lipid phase of transverse tubule membrane was probed with a variety of fatty acid spin labels. The motion of the probe increased as the distance between the spin label and polar head group increased, in agreement with results reported in other membranes. The value of the order parameter at 37 degrees C for a fatty acid spin label containing the label attached to its fifth carbon atom was closer to values reported for bacterial membranes than to the lower values reported for other mammalian membranes. Order parameters for spin labels containing the label nearer to the center of the bilayer were closer to the values reported in other mammalian membranes than to values reported for bacterial membranes. These results indicate that the lipid segments in the vicinity of the polar head group, and less so those near the center of the bilayer, are motionally more restricted in transverse tubules than in other mammalian membranes. In particular, the lipid phase of the transverse tubule membrane is less fluid than that of the sarcoplasmic reticulum membrane. A possible role of the high cholesterol content of transverse tubules in generating the lower fluidity of its lipid phase is discussed.     

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25 °C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.     

Comparison of membrane structure,osmotic fragility,and morphology of multiple sclerosis and normal erythrocytes

Erythrocyte membranes from multiple sclerosis (MS) patients and normal individuals were studied by electron spin resonance spectroscopy, osmotic fragility tests, scanning electron microscopy (SEM) and fatty acid analysis of membrane lipids. There was no significant difference in the membrane fluidity between MS and normal erythrocytes using fatty acid spin labels with the nitroxide moiety on carbons 5, 12, or 16 from the carboxyl group. Linoleic acid, which has been reported to decrease the absolute electrophoretic mobility of only MS erythrocytes, increased the fluidity of MS and normal erythrocyte membranes to a similar extent. The osmotic fragility of MS erythrocytes obtained from outpatients was similar to normal control cells but the osmotic fragility of erythrocytes obtained from hospitalized MS patients was greater than normal. Scanning electron microscopy of MS erythrocytes revealed no gross abnormalities. Cells incubated with linoleic acid had transformed from discocytes into sphero-echinocytes with prominent membrane surface indentations but MS and normal erythrocytes appeared identical. Of the fatty acid content of the total lipid extract, erythrocytes from most, but not all, MS hospitalized patients and some patients with other demyelinating diseases had relatively less (P<.001) 18:2 than the normal cells. These results indicate that at least some of the abnormalities reported in MS erythrocytes may only be found in hospitalized patients and may be due to other complications of the disease. They also indicate that the reported abnormal effects of linoleic acid on the electrophoretic mobility of MS erythrocytes may be caused by some other mechanism than an effect on the fluidity of the bilayer.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.     

Fluidity of human erythrocyte membrane and effect of chlorpromazine on fluidity and phase separation of membrane

The fluidity of human erythrocyte membrane, and the effect of chlorpromazine at prelytic and lytic concentrations on the fluidity have been studied by using three kinds of fatty acid spin labels and measuring the temperature dependence of Mg2+-ATPase activity. The Arrhenius plot of the apparent rotational correlation time, tau c, for probes I(12,3) and I(5,10) showed an abrupt discontinuity at about 30 degrees C, and the plot for I(1,14) at 25 degrees C, indicating that a large difference in the fluidity exists between the interior and the outer surface of the lipid bilayer. The portions of the fatty acid chain near the ten carbon bond lengths removed from the bilayer surface became more fluid by chlorpromazine treatment; there was a decrease in the break point to around 26 degrees C following treatment with 0.6 or 1 mM of the drug. Two breaks at 21 and 30 degrees C in the Arrhenius plot of the Mg2+-ATPase activity were observed in normal erythrocyte membrane. The activation energy of the Mg2+-ATPase reaction has the values of 3.0 and 22.1 kcal/mol above the upper break and below the lower break, respectively. The drug exposure induced only a slight shift in the break temperatures, while the treatment significantly enhanced the associated activation energies of the reaction. These results suggest that the boundary phospholipids of the Mg2+-ATPase in the membrane are probably more rigid than the bulk lipids.     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

Organization of membrane lipids and proteins in human En(a-) erythrocytes that lack the major sialoglycoprotein, glycophorin A. A spin-label study.

Membrane fluidity was studied by electron-spin-resonance techniques in human En(a-) erythrocytes that lack the major membrane sialoglycoprotein, glycophorin A. By using stearic acid spin labels with a doxyl group in the C-12 or C-15 positions, we demonstrated that the hydrophobic core in these cells was more fluid than in normal cells. Surface-located regions in isolated En(a-) membranes, when probed with stearic acid labelled in the C-5 position, appeared more stable than in normal membranes. In isolated En(a-) membranes, protein motion was decreased when probed with a nitroxide derivative of maleimide. After incubation with anti-(glycophorin A) antibodies protein motion and membrane fluidity were increased in normal membranes. This effect was observed also after spectrin depletion, which by itself increased protein motion but decreased membrane fluidity in the hydrophobic core of the membrane. The results show that membrane proteins influence the fluidity of membrane lipids.     

Motion of spin-labeled fatty acids in murine macrophages relation to cellular phagocytic activity

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels.Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings ($\text{2T}{}_{\mathrm{|}}$) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives.Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 °C, or by fixing them with 0.25 % glutaraldehyde.No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with $\text{cis-}$ or $\text{trans-}\text{unsaturated}$ fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.