Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Organization of lipids in sarcoplasmic reticulum membrane and Ca2+-dependent ATPase activity.

The organization of lipids in sarcoplasmic reticulum membrane was studied with a variety of stearic spin labels and a phosphatidylcholine spin label. The ESR spectra of the spin-labeled membranes consisted of two components, one due to labels in lipid bilayer structure and the other due to more immobilized labels. The relative intensity of the immobilized component increased when the lipid content of the membrane was decreased by treatment with phospholipase A [EC 3.1.1.4] and subsequent washing with bovine serum albumin. Membrane containing 30% of the intact phospholipid, i.e.0.15 mg of phospholipid per mg of protein, showed a spectrum consisting only of the immobilized component (the overall splitting ranged from 58.5 G to 60.5 G). The immobilized component was ascribed to lipids complexed with protein. The fraction of lipids in the two different organizations was determined from the ESR spectrum. The activity of the Ca2+-Mg2+ dependent ATPase [ATP phosphohydrolase, EC 3.6.1.3] was found to increase almost linearly with the lipid bilayer content in the membrane, whereas phosphoenzyme formation was almost independent of the bilayer content. This indicated that the bilayer structure is necessary for the ATPase to attain its full transport activity.     

Lipid--protein multiple binding equilibria in membranes

Phospholipids at the lipid--protein interface of membrane proteins are in dynamic equilibrium with fluid bilayer. In order to express the number of binding sites (N) and the relative binding constants (K) in terms of measurable quantities, the equilibrium is formulated as an exchange reaction between lipid molecules competing for hydrophobic sites on the protein surface. Experimental data are reported on two integral membrane proteins, cytochrome oxidase and (Na,-K)-ATPase, reconstituted into defined phospholipids. Electron spin resonance measurements on reconstituted preparations of beef heart cytochrome oxidase in 1,2-dioleoyl-sn-3-phosphatidylcholine containing small quantities of the spin-labeled phospholipid 1-palmitoyl-2-(14-proxylstearoyl)-sn-3-phosphatidylcholine (PC*) gave a linear plot of bilayer/bound PC* vs. the lipid/protein ratio as predicted by the theory, with K congruent to 1 and N = 40 (normalized to heme aa3). This demonstrates that the spin-label moiety attached to the hydrocarbon chain does not significantly perturb the binding equilibria. In the second experimental system, (Na,K)-ATPase purified from rectal glands of Squalus acanthias was reconstituted with defined phosphatidylcholines as the lipid solvent and spin-labeled phospholipids with choline or serine head groups (PC*, PS*) as the solute. The (Na,K)-ATPase has a larger number of lipid binding or contact sites (N = 60-65 per alpha 2 beta 2 dimer) and exhibits a detectably larger average binding constant for the negatively charged phosphatidylserine than for the corresponding phosphatidylcholine. These results show that a multiple equilibria, noninteracting site binding treatment can account for the behavior of lipids exchanging between the protein surface and the lipid bilayer. Selective sites among a background of nonselective sites are experimentally detectable as a change in the measured relative binding constant.     

Effects of potassium on lipid-protein interactions in light sarcoplasmic reticulum

This study shows the effect of K+ on phospholipid-protein interactions in light sarcoplasmic reticulum (LSR) as measured by 31P NMR. In the presence of 110 mM K+, a substantial effect of the membrane protein on the behavior of the phospholipids was detected. Subtracting the spectrum of the LSR lipid extract from the spectrum of the intact LSR membrane produced a difference spectrum of much greater breadth than the normal phospholipid bilayer powder pattern. This powder pattern is indicative of a phospholipid domain considerably more motionally restricted than the phospholipids in a normal phospholipid bilayer. The apparent axially symmetric powder pattern is consistent with axial diffusion. In a reconstituted membrane containing the calcium pump protein at a lipid/protein ratio much less than in the light sarcoplasmic reticulum, the broad component was more prominent. The relative resonance intensity of the broad component appeared to be proportional to the lipid/protein ratio of the membrane. In 10 mM K+, no broad powder pattern is observed in the corresponding difference spectrum. Thus, in the absence of potassium, the membrane protein has much less influence on the phospholipid of the membrane, as measured by 31P NMR. In addition to the effects of K+ on the membrane structure of the sarcoplasmic reticulum, K+ modulated the function of the calcium pump. The rate of calcium-dependent ATP hydrolysis increased in light sarcoplasmic reticulum when [K+] increased from 10 to 110 mM. The rate of calcium transport was also stimulated by an increase in K+.     

An electron spin resonance spin-label study of lipophilin in oriented phospholipid bilayers

Model membranes consisting of dimyristoyl phosphatidylcholine and a hydrophobic protein from bovine myelin, lipophilin, were studied using the cholesterol-resembling cholestane ESR spin label. Orientation of the membranes made it possible to deconvolute the spectra into two fractions, one of oriented spin labels reflecting phospholipid bilayer of high order, and one of isotropically tumbling spin labels ascribed to the lipid fraction surrounding the protein molecule (boundary lipid). This isotropic tumbling is different from the behavior of phospholipid molecules near the protein, which retain some degree of order, and indicates that the boundary lipid fraction in our model system forms a rather fluid environment for the protein. A nonlinear relation was found between protein concentration and amount of boundary spin labels. Addition of cholesterol decreases the amount of boundary spin labels. Both findings form evidence for a preferential binding of cholesterol by the membrane protein.     

Protein-induced formation of cholesterol-rich domains

A major protein of neuronal rafts, NAP-22, binds specifically to cholesterol. We demonstrate by circular dichroism that NAP-22 contains a significant amount of beta-structure that is not sensitive to binding of the protein to membranes, suggesting that a major portion of the protein does not insert deeply into the membrane. The free energy of binding of NAP-22 to liposomes of dioleoylphosphatidylcholine with 40% cholesterol is -7.3 +/- 0.5 kcal/mol. NAP-22 mixed with dipalmitoylphosphatidylcholine and 40% cholesterol partitions into the detergent insoluble fraction in the presence of 1% Triton X-100. NAP-22 also causes this insoluble fraction to become enriched in cholesterol relative to phospholipid, again demonstrating the ability of this protein to segregate cholesterol and phospholipids into domains. Differential scanning calorimetry results demonstrate that NAP-22 promotes domain formation in liposomes composed of cholesterol and phosphatidylcholine. This is shown by NAP-22-promoted changes in the shape and enthalpy of the phase transition of phosphatidylcholine as well as by the appearance of cholesterol crystallite transitions in membranes composed of phosphatidylcholine with either saturated or unsaturated acyl chains. In situ atomic force microscopy revealed a marked change in the surface morphology of a supported bilayer of dioleoylphosphatidylcholine with 0.4 mole fraction of cholesterol upon addition of NAP-22. Prior to the addition of the protein, the bilayer appears to be a molecularly smooth structure with uniform thickness. Addition of NAP-22 resulted in the rapid formation of localized raised bilayer domains. Remarkably, there was no gross disruption or erosion of the bilayer but rather simply an apparent rearrangement of the lipid bilayer structure due to the interaction of NAP-22 with the lipid. Our results demonstrate that NAP-22 can induce the formation of cholesterol-rich domains in membranes. This is likely to be relevant in neuronal membrane domains that are rich in NAP-22.     

Structural and dynamical aspects of membrane immunochemistry using model membranes.

Three different phospholipid haptens have been synthesized, in which the haptenic group is the paramagnetic nitroxide (spin-label) group. These lipid haptens differ from one another in the length and composition of the molecular chain linking the 2,2,6,6-tetramethylpiperidinyl-N-oxy moiety to the phosphodiester group of the lipid. These lipid haptens have been incorporated at low molar concentrations (0.01 to 0.5 mol %) in liposomes containing various proportions of cholesterol and dipalmitoylphosphatidylcholine (DPPC). A study has been made of specific antinitroxide IgG (and Fab) binding to these liposomes, and the fixation of complement. From these studies we conclude: (a) For lipid haptens whose possible extension above the bilayer plane is limited (e.g., approximately 10-20 A), antibody binding and complement fixation depend strongly on the hapten structure and host lipid composition, because of steric limitations on the accessibility of lipid haptens to the binding sites in the protein. (b) Complement fixation by specific IgG antibodies directed against the nitroxide group as part of a lipid hapten depends strongly on the lateral mobility of the lipid hapten when its molar concentration in the plane of the membrane is of the order of 0.1 mol % or less. It is likely that this conclusion applies to many lipid haptens, and possibly other membrane components. (c) The inclusion of cholesterol in lipid membranes has at least two distinct effects on complement fixation involving lipid haptens. Through a steric effect on bilayer structure (probably involving lateral molecular ordering) cholesterol in phosphatidylcholine bilayers can enhance hapten exposure to antibody binding sites, enhance antibody binding, and thereby enhance complement fixation. It is likely that cholesterol also affects complement fixation at low hapten concentrations through a modification of membrane fluidity.     

ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.     

Phosphatidylcholine exchange between the boundary lipid and bilayer domains in cytochrome oxidase containing membranes.

A phospholipid spin label, 16-doxylphosphatidylcholine, is employed in a study of lipid--protein interactions in cytochrome oxidase containing membranes. Two methods are used to label the membranous cytochrome oxidase: dispersion in cholate with subsequent detergent removal, and fusion with vesicles of the pure phospholipid label in the absence of detergent. A fraction of the label is immobilized, which is calculated to fall in the range of 0.17--0.21 mg of phospholipid/mg of protein (0.15--0.19 after correction for lipids not extracted by chloroform--methanol). This narrow range of values is independent of methods of labeling, protein isolation, and lipid depletion within experimental error. When labeling by fusion is utilized, the patches of pure phosphatidylcholine spin label diffuse in the plane of the bilayer, become diluted, and demonstrate exchange with bound phospholipid. These observations are evidence that boundary lipid, as reflected by the partitioning of the phosphatidylcholine label, is in equilibrium with adjacent bilayer regions and that it consists of a relatively constant amount of phospholipid associated with the hydrophobic portion of the protein.     

Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).     

Interactions of proteins and cholesterol with lipids in bilayer membranes.

Mixtures of lipids and protein, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-PO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, Tt, of the lipid and aggregated below Tt. For mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains were shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present. In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50-200 nm in length, around smooth patches of lipid. Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperature of the lipid is discussed.     

Interactions of cholesterol hemisuccinate with phospholipids and (Ca2+-Mg2+)-ATPase

Cholesterol hemisuccinate has been shown to equilibrate readily with liposomes and with the (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum and has been used to modify the sterol content of these membranes. Cholesterol hemisuccinate incorporates into dioleoylphosphatidylcholine (DOPC) up to a molar ratio of 3:1 sterol to DOPC. Effects on lipid order as detected by electron spin resonance and fluorescence polarization are comparable to those of cholesterol. Binding constants have been determined, and the uncharged form of the sterol binds more strongly than the anionic form. Binding to DOPC and to the lipid component of the ATPase system is comparable. From use of the fluorescence quenching properties of 1,2-bis(9,10- dibromooleoyl )phosphatidylcholine and dibromocholesterol hemisuccinate, two classes of binding sites on the ATPase have been deduced. At the lipid/protein interface, the binding constant for cholesterol hemisuccinate is considerably less than that for DOPC. At the second set of sites ( nonannular sites), binding occurs with Kd = 0.55 in molar ratio units. The effect of cholesterol hemisuccinate on the activity of the ATPase depends on the phospholipid present in the system: ATPase reconstituted with DOPC is inhibited whereas ATPase reconstituted with dimyristoleoylphosphatidylcholine is activated. We conclude that changes in membrane fluidity are not important in determining ATPase activity in these systems.     

Effects of lutein and cholesterol on alkyl chain bending in lipid bilayers: a pulse electron spin resonance spin labeling study.

A short pulse saturation recovery electron spin resonance technique has been used to study the effects of polar carotenoid-lutein and cholesterol on interactions of 14N:15N stearic acid spin-label pairs in fluid-phase phosphatidylcholine (PC) membranes. Bimolecular collisions for pairs consisting of various combinations of [14N]-16-, [14N]-10-, [14N]-7-, or [14N]-5-doxylstearate and [15N]-16-doxylstearate in dimyristoyl-PC (DMPC) or egg yolk PC (EYPC) membranes were measured at 27 degrees C. In the absence and presence of lutein or cholesterol for both lipid systems, the collision rates were ordered as 16:5 < 16:7 < 16:10 < 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in EYPC. Polar carotenoid-lutein reduces the collision frequency for all spin-label pairs, whereas cholesterol reduces the collision frequency for 16:5 and 16:7 pairs and increases the collision frequency in the membrane center for 16:10 and 16:16 pairs. The presence of unsaturated alkyl chains greatly reduces the effect of lutein but magnifies the effect of cholesterol in the membrane center. The observed differences in the effects of these modifiers on alkyl chain bending result from differences in the structure of cholesterol and polar carotenoid and from their different localization within the lipid bilayer membrane. These studies further confirm the occurrence of vertical fluctuations of alkyl chain ends toward the bilayer surface.     

Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers

Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Labelling of egg phosphatidylcholine vesicles and myelin membrane with a photoreactive lipophilic reagent.

The preparation and isolation of [3H]phenyl azide, a photosensitive non-polar probe, is reported. The reagent partitions into the lipid bilayer of egg phosphatidylcholine vesicles and bovine myelin membranes. On photoactivation to generate the nitrene grouping, as much as 90% of the covalently attached label is associated with the fatty acyl residues of the constituent phospholipid molecules. The remainder is found in the polar head groups. The cholesterol component of myelin membranes is also heavily labelled. These results suggest that such reagents may be used to probe the hydrophobic regions of natural membranes.     

The role of cholesterol in the activity of reconstituted Ca-ATPase vesicles containing unsaturated phosphatidylethanolamine

The effect of cholesterol on the activity of sarcoplasmic reticulum Ca-ATPase, reconstituted in proteoliposomes containing soybean phosphatidylethanolamine (PE), egg phosphatidylcholine (PC), and cholesterol, was examined. The protein incorporation efficiency increased with PE content but appeared to be independent of cholesterol content. At low cholesterol, PE stimulated calcium uptake. The coupling efficiency of the proteoliposomes increased with an increase in cholesterol content at each PC/(PC + PE) ratio and was more pronounced for those proteoliposomes containing high PE. Dynamic fluorescence measurements of the incorporated lipophilic probe, diphenyl-1,3,5-hexatriene, revealed a decrease in the motion and an increase in the order of the phospholipid fatty acyl chains in proteoliposomes with high cholesterol content. A complementary observation was made using electron spin resonance of the spin label, 2,2-dimethyl-5-dodecyl-5-methyloxazolidine N-oxide. Freeze-fracture electron microscopy studies on proteoliposomes containing 0.20 molar ratio of PC/(PC + PE) and cholesterol revealed predominantly vesicular structures with occasional bilayer defects at high cholesterol content. It is postulated that the cholesterol-induced enhancement of the Ca-transport function of the Ca-ATPase is related to the hydration-related bilayer-destabilizing characteristic of the cholesterol molecule as revealed by 31P NMR.     

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.     

The effects of cholesterol on lateral diffusion and vertical fluctuations in lipid bilayers. An electron-electron double resonance (ELDOR) study.

Electron-electron double resonance (ELDOR) and saturation-recovery spectroscopy employing 14N:15N stearic acid spin-label pairs have been used to study the effects of cholesterol on lateral diffusion and vertical fluctuations in lipid bilayers. The 14N:15N continuous wave electron-electron double resonance (CW ELDOR) theory has been developed using rate equations based on the relaxation model. The collision frequency between 14N-16 doxyl stearate and 15N-16 doxyl stearate, WHex (16:16), is indicative of lateral diffusion of the spin probes, while the collision frequency between 14N-16 doxyl stearate and 15N-5 doxyl stearate, WHex (16:5), provides information on vertical fluctuations of the 14N-16 doxyl stearate spin probe toward the membrane surface. Our results show that: (a) cholesterol decreases the electron spin-lattice relaxation time Tle of 14N-16 doxyl stearate spin label in dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (egg PC). (b) Cholesterol increases the biomolecular collision frequency WHex (16:16) and decreases WHex (16:5), suggesting that incorporation of cholesterol significantly orders the part of the bilayer that it occupies and disorders the interior region of the bilayer. (c) Alkyl chain unsaturation of the host lipid moderates the effect of cholesterol on both vertical fluctuations and lateral diffusion of 14N-16 doxyl stearate. And (d), there are marked differences in the effects of cholesterol on lateral diffusion and vertical fluctuations between 0-30 mol% and 30-50 mol% of cholesterol that suggest an inhomogeneous distribution of cholesterol in the membrane.     

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.     

Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms

Steroid hormones are lipophilic suggesting they intercalate into the bilayer of target cell plasma membranes, potentially altering the fluidity and function of the membrane. The present study measured the effects of steroidal exposure on both phospholipid fluidity and integral protein mobility. Studies were performed on the effects of a variety of steroids on phosphatidylcholine liposomes, synaptosomal plasma membranes and sarcoplasmic reticulum membranes. Progesterone decreased the lipid fluidity, whereas testosterone had no effect on lipid movement. The estrogen, 17 beta-estradiol, an aromatised metabolite of testosterone, increased lipid mobility. In each case, the steroid action was concentration-dependent. The steroids all increased the activity of the Ca2+ ATPase of SR membrane, in keeping with their effects on this enzyme's aggregation state. The results suggest that, although lipid fluidity is a factor influencing protein activity, their mobility within the bilayer is the primary determinant of enzyme activity in the membrane for most proteins.