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.     

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.     

A spin-label study of the chromaffin granule membrane.

The structure of the chromaffin granule membrane has been probed using a number of different spin labels. Both the effect of temperature and high levels of calcium have been studied. 1. The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which undergoes a structural transition at 32-36 degrees C, characterized by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol. 2. The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32 degrees C. This transition corresponds to an increased degree of motional freedom of the spin label above the transition temperature. 3. The lipid-soluble spin label 2,2,6,6-tetramethyl-piperidine-1-oxyl exhibits a break at 34 degrees C in the temperature-dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in the membrane lipid, again possible involving a re-distribution of cholesterol. 4. Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a redistribution of cholesterol and also lysolecithin. The temperatures of the structural transition agree well with the changes in the enzymic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al., Eur. J. Biochem. 67, 105-114(1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion.     

Model membrane studies of spin-label probes Part I. Mixed monolayers of 12-nitroxide stearic acid and myristic acid

Pure and mixed monomolecular films of a cell membrane spin label probe, 12-nitroxide stearic acid have been studied where myristic acid was selected as the host lipid. The behavior of 12-nitroxide stearic acid at the air water interface is understood in terms of two molecular configurations: erect (with only the carboxyl group in the interface) and bent (with both the carboxyl group and the oxazolidine ring in the interface). In mixed films both of these conformations play a role at high surface pressures. At low probe concentrations, 12-nitroxide stearic acid is primarily in an erect conformation, while at high probe concentrations the reverse is true. This particular host lipid appears capable of erecting the probe molecule with only small concentrations of myristic acid. In a condensed host lipid, the probe is partially immiscible, and segregates to form a heterogeneous film from which it is readily collapsed. The probe is seen to perturb the molecular packing in this mixed system and the perturbation to be dependent on both the molecular shape and nature of the probe.     

Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by parmagenetic lipid analogues. Introduction of a new probe approach.

1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.     

Electron spin resonance studies of the effects of lipids on the environment of proteins in mitochondrial membranes

The physical state of mitochondrial membranes has been investigated by means of stearic acid spin labels and of a maleimide spin label covalently bound to protein sulfhydryl groups. Stearic acid spin labels 5-NS and 16-NS show that n-butanol enhances the lipid fluidity of mitochondrial membranes in the whole temperature range between 4 and 37 degrees C; the effects in the hydrophobic membrane core, probed by 16-NS, are already apparent at 10 mM butanol. In liposomes formed of mitochondrial phospholipids, a fluidizing effect appears only at much higher concentration. Such results are compatible with the idea that butanol destabilizes lipid-protein interactions. On the other hand, the ratio between weakly and strongly immobilized SH groups probed by maleimide spin label is only slightly affected in the temperature range of 4-37 degrees C by addition of high concentrations of n-butanol, indicating that the environments probed are stable to agents inducing fluidity changes in the lipids. There are, however, indications that the environment probed by maleimide is affected by lipids, since the spin label, when bound to lipid-depleted mitochondria, becomes more immobilized, reconstitution of such lipid-depleted membranes with phospholipids restores the original spectra.     

Protein heat denaturation and study of membrane lipid-protein interactions by spin label ESR.

Spinach chloroplast membranes labelled with stearic acid-spin probe-bearing nitroxyl (label) moiety at 5th, 9th, 12th, 13th, 14th or 16th carbon locations with respect to the carboxylic group of stearic acid were studied (in the dark) by electron spin resonance (ESR) spectroscopy. Spectra were recorded at sample temperatures of 5, 30 and 67 degrees C. After heat denaturation of the membrane proteins for 5 min at 67 degrees C, the spectra were re-recorded at 30 and 5 degrees C for comparison. The results unequivocally show that membrane lipid fatty-acyl chains become substantially more rigid after protein heat-denaturation. The data throw light on the degree of lipid-protein interactions at various microlocations along the length of fatty-acyl chains of the membrane lipid matrix.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

Abnormal fluidity state in membranes of malignant hyperthermia pig skeletal muscle

The fluidity state was analyzed on sarcoplasmic reticulum membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the 5-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hypothesis for this syndrome.     

A 2H NMR study on alteration of the membrane organization of mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate

In order to monitor the membrane fluidity of cells without perturbation by an introduced probe, we developed a method for large-scale preparation of 2H-labeled melanoma cells for a 2H NMR study by incubating melanoma cells with [18,18,18-2H3]stearic acid/phosphatidylcholine liposomes for 2 h at 37 degrees C. It turned out that this treatment did not significantly change the cell viability, lipid metabolism or membrane fluidity. The 2H from C-18 of stearic acid is dominantly located at the original position of the fatty acid in the 2H-labeled membrane vesicles, as studied by a tracer experiment with [1-14C]stearic acid. We found that three to four 2H-labeled species were present at 19 degrees C in 2H NMR spectra of the 2H-labeled membrane vesicles prepared from B16 melanoma cells. The extent of peak-splittings due to 2H-quadrupole interaction decreased as the temperature rose, and a definite point of phase transition was not observed. At elevated temperature, 2H-labeled lipids undergo fast exchange between the bilayer and an isotropic phase such as oil phase of triolein or inverted micelles in lipid polymorphs. We further analyzed the change of membrane organization in mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), which strongly inhibited melanogenesis. The magnitude of the quadrupole splitting at 19 degrees C in membranes from TPA-treated cells was significantly less (40%) than in the untreated control. This is mainly explained by decreased molecular ordering (fluidity) due to the increased amount of unsaturated fatty acids in the membranes of TPA-treated cells.     

Electron spin resonance evidence for vertical asymmetry in animal cell membranes.

Two electron spin resonance (ESR) spin labels were used to monitor the physical state of bacterial and animal cell membranes: 5N10, a nitroxide derivative of decane, and 12NS-GA, a glucosamine derivative of 12-nitroxide stearic acid. Spectra were recorded at 1 degrees C intervals from approximately 5 to 45 degrees C. Arrhenius plots of log hH/hP vs. 1/K were obtained by measuring the amplitudes of the hydrocarbon and water signals, hH and hP, respectively. Two discontinuities in the Arrhenius plot (at characteristic temperatures t1 and th) were observed with bacterial cell membranes independent of the spin label employed. Analysis of sealed animal cell membrane samples revealed four characteristic temperatures when the hydrophobic spin lable 5N10 was used, but only two when the amphiphilic spin label 12NS-GA was used. The specific set of characteristic temperatures revealed with 12NS-GA depended on whether the membrane preparation was inside out (ISO) or right side out (RSO). Analysis of Newcastle disease virus, a source of RSO plasma membrane derived from host, revealed two characteristic temperatures at approximately 14 and 33 degrees C. Analysis of phagosomes, a source of ISO plasma membrane derived from LM cells, revealed two characteristic temperatures at approximately 23 and 38 degrees C. When unsealed or disrupted membrane preparations were spin labeled with 12NS-GA, both sets (RSO and ISO) of characteristic temperatures were revealed. The results indicate that the inner and outer monolayers of animal cell membranes are physically distinct and that the glycosylated spin label, 12NS-GA, is apparently restricted in its ability to flip across the membrane bilayer. In this study, characteristic temperatures were pinpointed by computer analysis of the ESR spectral data.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles.

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37 degrees C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20 degrees C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20 degrees C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freeze-thaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.     

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.     

Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.     

Characterization of thermotropic structural transitions of the erythrocyte membrane: a biochemical and electron-paramagnetic resonance approach

The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16-dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40 degrees C. The transition at 8 degrees C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20 degrees C phase transition is unmodified after chymotrypsin treatment and occurs at 15 degrees C after complete proteolysis of intramembrane chymotrypsin-insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15 degrees C. The 40 degrees C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low-ionic-strength resealed ghosts. Transitions at 8 degrees C and 40 degrees C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20 degrees C transition is intrinsic to the lipid component.     

Studies on spin-labelled egg lecithin dispersions.

ESR spectra of egg lecithin dispersions labelled with 5-nitroxide stearic acid are recorded with a 50 G field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The expansion technique improves the precision of the order parameters determined from the hyperfine splitting measurements, and may prove useful in future spin label membrane studies. Approximate order parameters are derived to describe the fluidity of fatty acid spin-labelled membranes in those cases where either the inner or outer hyperfine extrema are not well defined. The ability of these expressions to measure the fluidity of labelled egg lecithin disperions for the temperature range 14-42 degrees C is examined.     

A spin-label study of sciatic nerves from quaking, jimpy and trembler mice.

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant (T) between 0 degrees C and 50 degrees C. Within this range of temperatures, we obtained identical values of 2 T for nerves from normal and jimpy mice, whereas 2 T was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice. The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.     

Effect of high electrolyte concentration on the phase transition behaviour of DPPC vesicles: a spin label study

We have investigated by Electron Spin Resonance spectroscopy, the effects of high electrolyte concentration on the phase transitions of unilamellar vesicles of dipalmitoylphosphatidylcholine at the pH values of 5.0 and 9.0. Using the 5-nitroxide stearic acid as spin probe we have found that, at both pH values, the lipid main phase transition is not quite affected by variations of the electrolyte concentration up to the value of 3 M. Instead, the pretransition at pH 5.0 disappears in the presence of 1 M electrolyte, and at pH 9.0, the pretransition temperature shifts upward from 25.5 to 31.0 degrees C when the electrolyte concentration reaches the value of 3 M. The observed results on the pre- and main phase transition widths, transition temperatures and their cooperativity indicate that the presence of salt in the bulk solution leads to structural changes of the lipid bilayer which essentially concern either the polar zone or the hydrogen belt region of the DPPC vesicles. The extent of observed perturbation depends on salt concentration.     

Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.