The effect of bupivacaine·HCl on the physical properties of neuronal membranes

Fluorescent probe techniques were used to evaluate the effect of bupivacaine.HCl on the physical properties (transbilayer asymmetric lateral and rotational mobilities, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. An experimental procedure was used based on selective quenching of both 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer (RET) from the tryptophans of membrane proteins to Py-3-Py. Bupivacaine.HCl increased the bulk lateral and rotational mobilities, and annular lipid fluidity in SPMVs lipid bilayers, and had a greater fluidizing effect on the inner monolayer than that of the outer monolayer. The magnitude of increasing effect on annular lipid fluidity in SPMVs lipid bilayer induced by bupivacaine.HCl was significantly far greater than magnitude of increasing effect of the drug on the lateral and rotational mobilities of bulk SPMVs lipid bilayer. It also caused membrane proteins to cluster. These effects of bupivacaine.HCl on neuronal membranes may be responsible for some, though not all, of the local anesthetic actions of bupivacaine.HCl.     

The effect of ethanol on the physical properties of neuronal membranes

Intramolecular excimer formation of 1,3-di(1-pyrenyl) propane(Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to evaluate the effect of ethanol on the rate and range of lateral and rotational mobilities of bulk bilayer structures of synaptosomal plasma membrane vesicles (SPMVs) from the bovine cerebral cortex. Ethanol increased the excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py in the SPMVs. Selective quenching of both DPH and Py-3-Py by trinitrophenyl groups was used to examine the range of transbilayer asymmetric rotational mobility and the rate and range of transbilayer asymmetric lateral mobility of SPMVs. Ethanol increased the rotational and lateral mobility of the outer monolayer more than of the inner one. Thus ethanol has a selective fluidizing effect within the transbilayer domains of the SPMVs. Radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py was used to examine both the effect of ethanol on annular lipid fluidity and protein distribution in the SPMVs. Ethanol increased annular lipid fluidity and also caused membrane proteins to cluster. These effects on neuronal membranes may be responsible for some, though not all, of the general anesthetic actions of ethanol.     

Effects of chlorpromazine HCl on the structural parameters of bovine brain membranes

Fluorescence probes located in different membrane regions were used to evaluate the effects of chlorpromazine .HCl on structural parameters (transbilayer lateral mobility, annular lipid fluidity, protein distribution, and lipid bilayer thickness) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. The experimental procedure was based on the selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, radiationless energy transfer from the tryptophan of membrane proteins to Py-3-Py, and energy transfer from Py-3-Py monomers to 1-anilinonaphthalene-8-sulfonic acid (ANS). In this study, chlorpromazine .HCl decreased the lateral mobility of Py-3-Py in a concentration dependent-manner, showed a greater ordering effect on the inner monolayer than on the outer monolayer, decreased annular lipid fluidity in a dose dependent-manner, and contracted the membrane lipid bilayer. Furthermore, the drug was found to have a clustering effect on membrane proteins.     

Effects of dimyristoylphosphatidylethanol on the structural parameters of neuronal membranes

Fluorescent probes located in different membrane regions were used to evaluate the effects of dimyristoylphosphatidylethanol (DMPEt) on the structural parameters (transbilayer rotational and lateral mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) from the bovine cerebral cortex. An experimental procedure was used based on selective quenching of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. DMPEt increased the bulk lateral and rotational mobility, and annular lipid fluidity of SPMV lipid bilayers, and had a greater fluidizing effect on the outer monolayer than the inner monolayer. It also caused membrane proteins to cluster. These effects of DMPEt on neuronal membranes may be responsible for some, though not all, of the general anesthetic actions of ethanol.     

The effect of ethanol on lateral and rotational mobility of plasma membrane vesicles isolated from cultured mar 18.5 hybridoma cells

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to evaluate the effect of ethanol on the rate and range of the lateral mobility and the range of the rotational mobility of bulk bilayer structures of the plasma membrane vesicles (ATCC-PMV) isolated from cultured hybridoma cells (ATCC TIB 216). In a concentration-dependent manner, ethanol increased the excimer to monomer fluorescence intensity ratio (I/I) of Py-3-Py in the ATCC-PMV and decreased the anisotropy (r), limiting anisotropy (r) and order parameter (S) of DPH in the ATCC-PMV. This indicates that ethanol increased both the lateral and rotational mobility of the probes in the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was utilized to examine the range of transbilayer asymmetric rotational diffusion of the ATCC-PMV. The anisotropy (r), limiting anisotropy (r ) and order parameter (S) of DPH in the inner monolayer were 0.024, 0.032, and 0.069, respectively, greater than calculated for the outer monolayer of the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was also used to examine the transbilayer asymmetric effects of ethanol on the range of the rotational mobility of the ATCC-PMV. Ethanol had a greater increasing effect on the range of the rotational mobility of the outer monolayer as compared to the inner monolayer of the ATCC-PMV. It has been proven that ethanol exhibits a selective rather than nonselective fluidizing effect within the transbilayer domains of the ATCC-PMV.This paper was supported in part by a research grant from the Korea Science and Engineering Foundation (KOSEF 88-1013-01) and from the Korea Research Foundation (1991–1993).     

Effect of ionic polarizability on electrodiffusion in lipid bilayer membranes.

Ion-carrier complexes and organic ions of similar size and shape have mobilities in lipid bilayer membranes which span several orders of magnitude. In this communication, an examination is made of the hypothesis that the basis for this unusually wide range of ionic mobilities is the potential energy barrier arising from image forces which selectively act on ions according to their polarizability. Using Poisson's equation to evaluate the electrostatic interaction between an ion and its surroundings, the potential energy barrier to ion transport due to image effects is computed, with the result that the potential energy barrier height depends strongly on ionic polarizability. Theoretical membrane potential energy profile calculations are used in conjunction with Nernst-Planck electrodiffusion equation to analyze the available mobility data for several ion-carrier complexes and lipid-soluble ions in lipid bilayer membranes. The variation among the mobilities of different ions is shown to be in agreement with theoretical predictions based on ionic polarizability and size. Furthermore, the important influence exerted by image forces on ion transport in lipid bilayer membranes compared to the frictional effect of membrane viscosity is established by contrasting available data on the activation energy of ionic conductivity with that for membrane fluidity.     

Lipid peroxidation-induced changes in physical properties of annular lipids in rat brain synaptosomal membranes

The effects of lipid peroxidation (LPO) on the physical state (fluidity) of the rat brain synaptosomal lipid bilayer matrix and the annular lipid domains were investigated using the fluorescent probe pyrene. The parameters of pyrene fluorescence intensity alpha = IE/IM were measured at excitation wavelengths 280 nm and 340 nm (alpha 280 and alpha 340), reflecting fluidity of lipid bilayer matrix and annular lipids, respectively. LPO induction was shown to result in changes of fluidity of both the bilayer and annular lipids. Upon reducing formation of LPO products by carnosine, fluidity changes of both the lipid bilayer matrix and annular lipids were diminished. Conformational changes of the annular lipid domain by LPO may therefore be considered as a possible cause of the functional changes in the receptor mediated responses and of the inactivation of membrane-bound enzymes by oxidative stress.     

Aluminum affects membrane physical properties in human neuroblastoma (IMR-32) cells both before and after differentiation

The capacity of Al(3+) to induce changes in the physical properties of plasma membrane from human neuroblastoma cells (IMR-32) was investigated, and the magnitude of the changes was compared with that obtained after cell differentiation to a neuronal phenotype. Similarly to our previous results in liposomes, Al(3+) (10 to 100 microM) caused a significant loss of membrane fluidity, being the differentiated cells more affected than the nondifferentiated cells. Al(3+) also increased the relative content of lipids in gel phase and promoted lipid rearrangement through lateral phase separation, with the magnitude of this effect being similar in nondifferentiated and differentiated cells. Since membrane physical properties depend on bilayer composition, we characterized the content of proteins, phospholipids, cholesterol, and fatty acids in the IMR-32 cells before and after differentiation. Differentiated cells had a significantly higher content of unsaturated fatty acids, creating an environment that favors Al(3+)-mediated effects on the bilayer fluidity. The neurotoxic effects of Al(3+) may be, at least in part, due to alterations of neuronal membrane physical properties, with potential consequences on the normal functioning of membrane-related cellular processes.     

The Fluid–Mosaic model of cell membranes: A brief introduction,historical features,some general principles,and its adaptation to current information

The Fluid–Mosaic Membrane (FMM) model was originally proposed as a general, nanometer-scale representation of cell membranes (Singer and Nicolson, 1972). The FMM model was based on some general principles, such as thermodynamic considerations, intercalation of globular proteins into a lipid bilayer, independent protein and lipid dynamics, cooperativity and other characteristics. Other models had trimolecular structures or membrane globular lipoprotein units. These latter models were flawed, because they did not allow autonomous lipids, membrane domains or discrete lateral dynamics. The FMM model was also consistent with membrane asymmetry, cis- and trans-membrane linkages and associations of membrane components into multi-molecular complexes and domains. It has remained useful for explaining the basic organizational principles and properties of various biological membranes. New information has been added, such as membrane-associated cytoskeletal assemblies, extracellular matrix interactions, transmembrane controls, specialized lipid-protein domains that differ in compositions, rotational and lateral mobilities, lifetimes, functions, and other characteristics. The presence of dense, structured membrane domains has reduced significantly the extent of fluid-lipid membrane areas, and the FMM model is now considered to be more mosaic and dense than the original proposal.     

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.     

Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius.

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.     

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance.

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.     

Effects of docosahexaenoic acid on annular lipid fluidity of the rat bile canalicular plasma membrane.

The role of docosahexaenoic acid (DHA) in the fluidity of the annular lipid regions and their associated membrane-bound proteins is still not as well understood as that in the global (bulk) lipid regions. We therefore studied the effects of dietary DHA on the relationship between annular and global lipid fluidity and membrane-bound enzymes such as 5'-nucleotidase and Mg(2)+-ATPase in the rat bile canalicular membrane. Dietary DHA caused significant increases in 5'-nucleotidase and Mg(2)+-ATPase activity and in global and annular lipid fluidity, a higher increase in fluidity in the annular lipids than the global lipids, and a decrease in the cholesterol-to-phospholipid molar ratio in the canalicular membrane. Plasma total cholesterol and LDL cholesterol decreased, and fecal cholesterol increased in the DHA-fed rats. No changes were observed in oxidative markers, but glutathione peroxidase increased in the liver with DHA feeding. Annular lipid fluidity, but not global lipid fluidity, correlated remarkably well with DHA, synchronously with the activities of 5'-nucleotidase and Mg(2)+-ATPase. The data indicate that the DHA-induced increase in annular lipid fluidity is responsible for the increases observed in the enzyme activity. We therefore concluded that the increased activity of membrane-bound enzymes and transporters induced by DHA and the concomitant increase in annular lipid fluidity comprise one of the mechanisms involved in DHA-induced clearance of plasma cholesterol.     

Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes.Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes.Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system.The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique.Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.     

Orientation and motion of spin-labels in rabbit small intestinal brush border vesicle membranes

The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as a bilayer. Compared to other mammalian membranes, the brush border membrane appears to be characterized by a relatively high packing order as well as microviscosity. At body temperature, the lipid molecules undergo rapid, anisotropic motion, which is essentially a fast rotation about an axis approximately perpendicular to the bilayer normal. Both the order (motional anisotropy) and the microviscosity increase with decreasing temperature and with increasing distance from the center of the bilayer. Qualitatively similar motional or fluidity gradients have been reported for other mammalian and bacterial membranes. The liposomes made from the extracted lipids have a somewhat lower packing order and a slightly higher fluidity than brush border vesicle membranes. The differences are, however, small indicating that the packing and the fluidity (microviscosity) of the membrane are primarily determined by the lipid composition. Membrane-associated proteins and cytoskeleton cannot play a dominant role in determining the order and fluidity of the lipid bilayer. Discontinuities are observed in the temperature dependence of various spectral parameters, the order parameter S, the rotational correlation time tau, and 2,2,6,6-tetramethylpiperidinyloxy partitioning. They are assigned to phase transitions and/or phase separations of the membrane lipids. These discontinuities occur at about 30, 20, and 13 degrees C for 5-doxyl-, 12-doxyl-, and 16-doxylstearic acid, respectively. The apparent transition temperature depends on the location of the spin probe along the bilayer normal, being higher the closer the probe is to the membrane surface. This indicates the possibility that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward.     

Effect of the functional groups of tocopherol molecules on lipid viscosity of mitochondria

The influence of tocopherol and its analogue (oxychroman) on the microviscosity of mitochondrial lipids was studied, using spin labels. The viscosity of the lipid bilayer was shown to enhance with the increase in the antioxidant content in the membrane. Small concentrations of alpha-tocopherol (10(-5)-10(-6) mol/l) were shown to increase, while large concentrations (10(-3)-10(-4) mol/l) decreased the fluidity of the lipid bilayer. The influence of alpha-tocopherol on fluidity of the lipid bilayer depending on its concentration could be realized in two ways: by direct influence on the lipid bilayer and via reception. It was shown that alterations in the viscosity of the lipid bilayer depend on chroman cycle of tocopherols, while the temperature of structural transfer and effective energy of activation depend on the lateral phytyl chain.     

Lateral mobility of phospholipids in the external and internal leaflets of normal and hereditary spherocytic human erythrocytes

The lateral diffusion coefficients (D) and the mobile fractions of the fluorescent phospholipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) and of membrane proteins labelled with fluorescein isothiocyanate, were measured by fluorescence photobleaching recovery on erythrocytes from healthy persons and from a hereditary spherocytosis patient. Measurements of lipid probe mobility were performed on ghosts labelled by NBD-PE exclusively at the external monolayer, or at both sides of the membrane. Our results indicate the following: (1) The mean values and the temperature dependence of D are different at the external and internal membrane leaflets. (2) In both normal and HS ghosts the mobile fraction of NBD-PE in the external monolayer does not depend significantly on temperature. On the other hand, the mobile fraction in the internal monolayer is reduced as the temperature is decreased. (3) At low temperatures, the mobile fraction of NBD-PE in the internal monolayer of spherocytic ghosts is significantly lower than the mobile fraction in the internal monolayer of normal ghosts. (4) No differences were observed between the mobilities of membrane proteins in normal and in spherocytic ghosts. However, differences were observed between the two cell populations in the temperature-dependence of the intrinsic fluorescence of unlabelled membrane proteins. The implications of these results for membrane phospholipid asymmetry and for cytoskeletal interactions with the internal lipid monolayer are discussed.     

Effect of Gold Nanoparticle on Structure and Fluidity of Lipid Membrane

This paper deals with the effect of different size gold nanoparticles on the fluidity of lipid membrane at different regions of the bilayer. To investigate this, we have considered significantly large bilayer leaflets and incorporated only one nanoparticle each time, which was subjected to all atomistic molecular dynamics simulations. We have observed that, lipid molecules located near to the gold nanoparticle interact directly with it, which results in deformation of lipid structure and slower dynamics of lipid molecules. However, lipid molecules far away from the interaction site of the nanoparticle get perturbed, which gives rise to increase in local ordering of the lipid domains and decrease in fluidity. The bilayer thickness and area per head group in this region also get altered. Similar trend, but with different magnitude is also observed when different size nanoparticle interact with the bilayer.     

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S(2)) and deuterium (S(CD)) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10-20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.     

Differential effect of lipid peroxidation on membrane fluidity as determined by electron spin resonance probes

The effect of lipid peroxidation on membrane fluidity was examined in sonicated soybean phospholipid vesicles. Following iron/ascorbate dependent peroxidation, the vesicles were labeled with a series of doxyl stearate spin probes which differed in the site of attachment of the nitroxide free radical to the fatty acid. Comparison of motional and partitioning parameters derived from electron spin resonance spectra of the probes indicated that the membranes were less fluid following peroxidation. However, the magnitude of the fluidity decrease was markedly dependent on the intramembrane location, as well as on the extent of lipid peroxidation. The effect of lipid peroxidation on fluidity was maximal in the membrane microenvironment sampled by 12-doxyl stearate, whereas other regions of the bilayer were less affected. These findings indicate that lipid peroxidation leads to an alteration of the transbilayer fluidity gradient.