Transbilayer Distribution of Cholesterol Is Modified in Brain Synaptic Plasma Membranes of Knockout Mice Deficient in the Low-Density Lipoprotein Receptor, Apolipoprotein E, or Both Proteins

Abstract: Both apolipoprotein E (apoE) and the low-density lipoprotein (LDL) receptor are present in brain; however, little is known regarding the function of these proteins in brain, in particular with respect to brain cholesterol. The role of apoE and the LDL receptor in modulating the transbilayer or asymmetric distribution of cholesterol in the exofacial and cytofacial leaflets of synaptic plasma membranes (SPMs) was examined in mutant mice deficient in apoE, the LDL receptor, or both proteins by using the fluorescent sterol dehydroergosterol and fluorescent quenching procedures. Fluidity of the exofacial and cytofacial leaflets was also measured. Cholesterol asymmetry of SPMs was altered in the mutant mice, with the largest effect observed in the LDL receptor-deficient mice. There was an approximately twofold increase in the percent distribution of cholesterol in the exofacial leaflet of the LDL receptor-deficient mice (32%) compared with C57BL/6J mice (15%). Mice deficient in apoE or both proteins also showed a significantly higher percent distribution of cholesterol (23 and 26%, respectively) in the exofacial leaflet compared with the C57BL/6J mice. Although the percent distribution of cholesterol was highest in the exofacial leaflet of the LDL receptor-deficient mice, fluidity of the exofacial leaflet of that group was significantly lower. However, the cholesterol-to-phospholipid ratio of SPMs of the LDL receptor-deficient mice was significantly lower, and this difference was largely the result of a significant increase in the total amount of SPM phospholipid. This study demonstrates for the first time that SPM lipid structure is altered in mice deficient in apoE or the LDL receptor. Although the mechanism that maintains the asymmetric distribution of cholesterol in plasma membranes is not well understood, data of the present experiments indicate that both apoE and the LDL receptor are involved in maintaining the transbilayer distribution of cholesterol.     

Acute and Chronic Effects of Ethanol on Transbilayer Membrane Domains

Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol-treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol-treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.     

Cholesterol asymmetry in synaptic plasma membranes

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function.     

Role of polyunsaturated fatty acids and lipid peroxidation in LM fibroblast plasma membrane transbilayer structure

The effects of polyunsaturated fatty acids and lipid peroxidation on LM fibroblast plasma membrane individual leaflet sterol distribution and structural order were examined. The cytofacial (inner) leaflet was more rigid and contained more sterol than the exofacial (outer) leaflet. The static (limiting anisotropy) and dynamic (rotational relaxation time) structural components of diphenylhexatriene (DPH) motion in each leaflet were determined by phase and modulation fluorometry measurements combined with leaflet-specific quenching by trinitrophenyl groups. Polyunsaturated fatty acids, incorporated into the membrane phospholipids by culture medium supplementation, decreased the limiting anisotrophy of DPH in the cytofacial but not the exofacial leaflet thereby abolishing the transbilayer difference in fluidity. Peroxidation by Fe(II) + H2O2 resulted in a rigidification (increase in limiting anisotropy and rotational relaxation time) of the plasma membrane exofacial leaflet, regardless of whether the membranes contained saturated and monounsaturated fatty acids or were enriched in either linoleate or linolenate. The structure of the cytofacial leaflet reported by DPH was unaffected. Plasma membrane transbilayer sterol distribution, measured by leaflet-specific quenching of dehydroergosterol fluorescence, indicated that 20-28% of the sterol was localized in the exofacial leaflet. Polyunsaturated fatty acid supplementation of LM fibroblasts resulted in a complete reversal of plasma membrane transbilayer sterol distribution (72-76% exofacial leaflet). Sterol transbilayer distribution between the membrane leaflets was completely resistant to alteration by exposure to crosslinking agents and peroxidation in control plasma membranes and by peroxidation in linoleate- or linolenate-supplemented membranes.     

Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets.

The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5'-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.     

Asymmetric distribution of a fluorescent sterol in synaptic plasma membranes: effects of chronic ethanol consumption

Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85-94) and in vivo ((1989) J. Neurochem. 52, 1925-1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids.     

Differential modulation of human small intestinal brush-border membrane hemileaflet fluidity affects leucine aminopeptidase activity and transport of D-glucose and L-glutamate.

The fluidity of the exofacial (outer) and cytofacial (inner) leaflets of human proximal small intestinal brush-border membrane vesicles was studied by selective quenching by trinitrophenyl groups, steady-state fluorescence polarization, and differential polarized phase fluorometry techniques, utilizing the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Differences in the hemileaflet's phospholipid composition were also analyzed by trinitrophenylation of aminophospholipids and phospholipase A2 treatment of these preparations. The results of these studies demonstrated that the inner leaflet of these membranes was less fluid than its outer counterpart. Phosphatidylserine was located mainly in the inner hemileaflet, whereas phosphatidylethanolamine and phosphatidylcholine were more symmetrically distributed between the hemileaflets of this membrane. Moreover, in vitro addition of 2-[(2-methoxyethoxy)ethyl]-cis-8-(2-octylcyclopropyl)octanoate (final concentration, 7.5 microM) preferentially fluidized the cytofacial leaflet and concomitantly increased Na(+)-gradient-dependent D-glucose uptake, but decreased Na+, K+-dependent L-glutamic acid uptake in these membrane vesicles. In vitro addition of benzyl alcohol (final concentration, 25 mM) preferentially fluidized the exofacial leaflet and decreased leucine aminopeptidase activity in these preparations. These results, therefore, demonstrate that the hemileaflets of human small intestinal brush-border membranes have different phospholipid compositions and fluidities. Alterations of either the exofacial or cytofacial leaflet fluidity, moreover, modulate protein-mediated activities in a distinct manner.     

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

Charged anesthetics selectively alter plasma membrane order

Although indirect evidence supporting differential lipid fluidity in the two monolayers of plasma membranes has accumulated, unambiguous demonstration of this difference has been difficult to obtain. In the present study, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), selective quenching of fluorescence by trinitrophenyl groups, and differential polarized phase fluorescence techniques were used to directly examine the static (order) and dynamic (rotational rate) components of lipid motion in the exofacial and cytofacial leaflets of LM fibroblast plasma membranes. The limiting anisotropy (0.137), the order parameter (0.590), and the rotational relaxation time (1.20 ns) of DPH in the plasma membranes (inner plus outer leaflet) indicated rapid but restricted probe motion in the lipid environment. However, the statics and dynamics of DPH motion in the individual monolayers were significantly (p less than 0.025) different. The limiting anisotropy, order parameter, and rotational relaxation time of DPH in the cytofacial monolayer were 0.036, 0.08, and 0.16 ns, respectively, greater than calculated for the exofacial monolayer of the LM plasma membrane. At appropriate concentrations, phenobarbital and, to a lesser degree, pentobarbital preferentially reduced the limiting anisotropy of DPH calculated for the exofacial leaflet while prilocaine reduced the limiting anisotropy of DPH in the cytofacial leaflet of LM fibroblast plasma membranes. In contrast, the putative cytofacial anesthetic procaine failed to show any preference for either leaflet. Arrhenius plots of DPH fluorescence in LM plasma membranes showed a prominent characteristic break point near 30-32 degrees C. Phenobarbital, pentobarbital, and procaine did not affect this break point while prilocaine selectively abolished it. The break point was therefore assigned to the inner monolayer of the LM plasma membrane.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.     

Transbilayer effects of ethanol on fluidity of brain membrane leaflets

Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.     

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to min, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate- or linolenate-supplemented medium, 71.8% and 75.5% of the plasma membrane sterol is exofacial, respectively.     

An InCytes from MBC Selection: Sterols Are Mainly in the Cytoplasmic Leaflet of the Plasma Membrane and the Endocytic Recycling Compartment in CHO Cells

The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15–32%. When the same quenchers have access to both leaflets, they quench 70–80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60–70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes ∼40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation.     

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.     

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.     

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.     

Effects of Age, Water Stress, and 1-Aminocyclopropane-1-carboxylic Acid on Leaflet Movement in Albizzia julibrissin

The movement patterns of Albizzia julibrissin leaflets transferred from light to darkness differ for leaflets of different age: the older the leaflet, the slower and less completely it closes. Water stress, which enhances ethylene synthesis in other plants, and 1-aminocyclopropane-1-carboxylic acid (ACC), precursor of ethylene, both mimic the effect of aging by reducing the rate and extent of dark-induced closure. Brief far-red compared to red irradiation before darkness does not appear to alter the closure of young leaflets, but far-red preirradiation retards the closure of middle-aged and old leaflets floating on water, and middle-aged leaflets treated with ACC. A change in some membrane property and/or cell wall lignification are suggested as possible explanations for the alteration of leaflet movement.     

Amyloid β-Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β-peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ_1–40 and Aβ_25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly ( p ≤ 0.02) by Aβ_1–40. Similar effects on fluidity were observed for Aβ_25–35 ( p ≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased ( p ≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.     

n-Amyl alcohol partitioning in synaptic plasma membranes

Nuclear magnetic resonance and fluorescence polarization techniques were used to determine n-amyl alcohol partitioning between, and effects on, lipid microdomains of isolated rat cerebral synaptic plasma membranes, n-Amyl alcohol binding to the hydrophobic membrane core had an unchanging binding constant over an aqueous alcohol concentration range of 2.5–22.5 mM, indicating a linear relationship between membrane core and aqueous alcohol concentrations. Binding to the membrane surface, in contrast, was cooperative with a steadily increasing binding constant over this alcohol concentration range. Membrane lipid order was determined using various fluorescent probes with preferences for the membrane core, for the mid-acyl regions of the exofacial or cytofacial bilayer leaflets and for ordered or bulk microdomains. All these probes showed steady decreases in membrane order with increasing alcohol concentration, at least for the nanosecond time scale sampled by this technique. These results further demonstrate the complexity of interaction between natural membranes and membrane disordering agents.