Fluid-phase Membranes Research Articles

Association and release of prostaglandin E 1 from liposomes

PGE1–lipid interactions were studied in several liposome systems. Data from both circular dichroic (CD) measurements and differential scanning calorimetry (DSC) indicated that PGE1 in the protonated form seeks the less polar environment of the lipid bilayer. CD measurements made on PGE1 in solution showed that the wavelength of maximum absorbance red shifted approximately 8 nm with decreasing solvent polarity. The CD spectrum of liposomal PGE1 prepared in pH 4.5 but not pH 7.2 buffer was also red shifted. There was no red shift in the CD spectrum of PGE1 detected at pH 4.5 in the absence of phospholipid. DSC measurements on DSPC bilayers prepared with 5 mol% PGE1 at pH 4.5 but not pH 7.2 revealed an almost complete loss of the pre-transition as well as broadening of the main phase transition. The amount of 3H-PGE1 initially associated with EPC, POPC or DSPC liposomes was determined using size exclusion filters and centrifugation. This amount was found to be dependent on the pH of the buffer (pH 4.5≫pH 7.2) and fluidity of the bilayer (EPC=POPC>DSPC), but independent of the lamellarity of the liposome. In all cases, addition of cholesterol reduced the amount of PGE1 associated with the liposome. The time-dependent release of PGE1 from the liposomes was determined by rapidly diluting the sample 100-fold into pH 7.2 buffer. Lipid saturation was a key factor influencing this release. Gel-phase liposomes of DSPC showed a rapid initial release ( t 1/2<2 min) of PGE1, corresponding to the amount in the outer monolayer, followed by a very slow, almost negligible release of the remaining PGE1. A rapid initial release also occurred in fluid-phase membranes, followed by a more gradual release of the remaining PGE1 over several hours. This release rate could be slowed by increasing the lamellarity of these liposomes, or adding cholesterol to decrease the fluidity of the membrane.

Thermal shape fluctuations of fluid-phase phospholipid-bilayer membranes and vesicles

After an introductory review of the physics of lipid-bilayer self-assembly, we discuss the origins of the area-difference elasticity model, a Landau-like theory which describes the mesoscale shape mechanics of bilayer sheets and vesicles. We introduce the notion of a phase diagram for mechanically stable low-energy vesicle shapes. We summarize what is now known about these shapes and about the transitions between them induced by changing control parameters, especially emphasizing the importance of metastability. In a final section devoted to recent work on thermal fluctuations, we outline a program for calculating normal mode energies and eigenshapes.

Structural characterization of free and membrane-bound nisin by infrared spectroscopy

This study reports two new trends about nisin affinity for lipid membranes. First, there is a very strong dependence of nisin binding on the membrane surface charge. As illustrated in this work, the binding of nisin is much greater for phosphatidylglycerol (PG) than for phosphatidylcholine (PC) membranes. This can be rationalized by electrostatic attraction between the positively charged peptide and the negatively charged PG. Second, the affinity of nisin shows a very weak dependence on the lipid phase, the binding to fluid or gel phase membranes being nearly equivalent. Therefore, our results suggest that nisin behaves as an extrinsic peptide. This work also presents the first piece of information relative to the structure of membrane-bound nisin. The Amide I band of the peptide is different for free nisin in water and for membrane-bound nisin. By analyzing this region using self-deconvolution and band fitting, and by comparing with results obtained from nisin dissolved in various H 2O/trifluoroethanol mixtures, it can be inferred that the binding of nisin to phospholipid membranes leads to an increased proportion of β-turns.

Permeability of nitric oxide through lipid bilayer membranes.

Profiles of the local nitric oxide (.NO) diffusion-concentration product across the egg yolk phosphatidylcholine membrane in the absence and presence of 30 mol% cholesterol were obtained using line-broadening electron paramagnetic resonance (EPR) and lipid-soluble nitroxide spin labels. Membrane .NO permeability coefficients were calculated from these profiles. At 20 degrees C, values of 93 and 77 cm/s for membranes in the absence and presence of cholesterol were obtained, compared with 73 and 66 cm/s for water layers of the same thickness as the membranes. Fluid-phase membranes are not barriers to .NO transport. Cholesterol significantly increases .NO transport in the center of the lipid bilayer.

Cholesterol content of trout plasma membranes varies with acclimation temperature.

Involvement of cholesterol in thermally induced restructuring of biological membranes was investigated in several tissues of rainbow trout (Oncorhynchus mykiss). Cholesterol-rich plasma membranes (PM) were isolated from erythrocytes, liver, kidney, and gill of fish acclimated to 5 and 20 degrees C. Mean PM cholesterol-to-phospholipid molar ratios (C/P) from warm-acclimated animals were significantly higher than those of cold-acclimated fish in liver (0.26 vs. 0.18; P < 0.01), kidney (0.49 vs. 0.40; P < 0.02), and gill (0.66 vs. 0.60; P < 0.05); erythrocyte C/P did not differ significantly with acclimation temperature (0.28 vs. 0.25; P = 0.25). In light of the ordering effects of cholesterol on fluid-phase membranes, these results are consistent with a role for cholesterol in the homeoviscous response of some poikilotherm PMs. Tissue differences in both PM cholesterol levels and the magnitude of thermally evoked cholesterol changes may reflect tissue-specific membrane functions. Lower PM C/P of trout tissues relative to corresponding data available for homeotherms also support a possible evolutionary relationship between cholesterol content and thermal adaptation of the PM.

Effects of probucol on phase transition and fluidity of phosphatidylcholine membranes: A spin label study

Spin labeling methods were applied to study the structure and dynamics of phosphatidylcholine membranes as a function of temperature and the mole fraction of probucol. Multilamellar liposomes made of dimyristoylphosphatidyclcholine, dipalmitoylphosphatidylcholine both saturated, and egg yolk phosphatidylcholine, an unsaturated membrane, were used. In fluid phase membranes probucol was found to increase the order and decrease the motional freedom of alkyl chains of lipids as shown with stearic acid spin labels. The effect of probucol on order and motional freedom is more pronounced in the membrane center (16-doxylstearic acid spin label position) than in the near polar headgroup region (5-doxylstearic acid spin label position). The presence of unsaturation in alkyl chains significantly decreased the ordering effect of probucol. The main phase transition temperature of saturated bilayers was lowered by 2°C in the presence of 3 mol% of probucol and significantly broadened at higher concentrations as measured with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) partitioning. Also, pretransition was no longer observed in the presence of probucol. In gel phase membranes, the effect of probucol was complex. Close to the main phase transition the motion of alkyl chains was increased, showing a regulatory effect of probucol on membrane fluidity. It is proposed that probucol is located in the membrane center as opposed to vitamin E, which locates its phenolic -OH group at the membrane surface; therefore, it inhibits lipid peroxidation in this region which is less accessible to vitamin E.

Hydrophobic Barriers of Lipid Bilayer Membranes Formed by Reduction of Water Penetration by Alkyl Chain Unsaturation and Cholesterol

The hydrophobicity profiles across phosphatidylcholine (PC)-cholesterol bilayer membranes were estimated in both frozen liposome suspensions and fluid-phase membranes as a function of alkyl chain length, unsaturation, and cholesterol mole fraction. A series of stearic acid spin labels, with the probe attached to various positions along the alkyl chain, cholesterol-type spin labels (cholestane and androstane spin labels), and Tempo-PC were used to examine depth-dependent changes in local hydrophobicity, which is determined by the extent of water penetration into the membrane. Local hydrophobicity was monitored primarily by observing the z component of the hyperfine interaction tensor (Az) of the nitroxide spin probe in a frozen suspension of the membrane at -150 degrees C and was further confirmed in the fluid phase by observing the rate of collision of Fe(CN)6(3-) with the spin probe in the membrane using saturation recovery ESR. Saturated-PC membranes show low hydrophobicity (high polarity) across the membrane, comparable to 2-propanol and 1-octanol, even at the membrane center where hydrophobicity is highest. Longer alkyl chains only make the central hydrophobic regions wider without increasing the level of hydrophobicity. Introduction of a double bond at C9-C10 decreases the level of water penetration at all locations in the membrane, and this effect is considerably greater than the cis configuration than with the trans configuration. Incorporation of cholesterol (30 mol %) dramatically changes the profiles; it decreases hydrophobicity (increases water penetration) from the polar headgroup region to a depth of approximately C7 and C9 for saturated- and unsaturated-PC membranes, respectively, which is about where the bulky rigid steroid ring structure of cholesterol reaches in the membrane. Membrane hydrophobicity sharply increases at these positions from the level of methanol to the level of pure hexane, and hydrophobicity is constant in the inner region of the membrane. Thus, formation of effective hydrophobic barriers to permeation of small polar molecules requires alkyl chain unsaturation and/or cholesterol. The thickness of this rectangular hydrophobic barrier is less than 50% of the thickness of the hydrocarbon regions. Results obtained in dioleoyl-PC-cholesterol membranes in the fluid phase are similar to those obtained in frozen membranes. These results correlate well with permeability data for water and amino acids in the literature.

Spin-label studies on phosphatidylcholine-polar carotenoid membranes: effects of alkyl-chain length and unsaturation

Spin-labeling methods were used to study the structure and dynamic properties of phosphatidylcholine (PC)-dihydroxycarotenoid membranes as a function of phospholipid alkyl chain length, alkyl chain saturation, temperature and mol fraction of carotenoids. (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin increase order and decrease motional freedom of the lipid alkyl chains in fluid-phase PC membranes. The effect of carotenoids decreases as the alkyl chain length of saturated PC increases. (2) The abrupt changes of spin-label motion observed at the main-phase transition of the saturated PC membranes are broadened and shifted to lower temperatures. At a carotenoid concentration of 10 mol%, they disappear for short-chain PC membranes (12–14 carbons), but are still observed for long-chain PC membranes (18–22 carbons). (3) In fluid-phase PC membranes possessing short alkyl chains (12–14 carbons), the activation energy of the rotational diffusion of 16-doxylstearic acid spin label (16-SASL) is significantly lower at a carotenoid concentration of 10 mol%. The difference decreases as the alkyl-chain length increases. (4) The presence of unsaturated alkyl chains greatly reduces the effects of carotenoids on the mobility of the polar headgroups as observed with tempocholine dipalmitoylphosphatidic acid ester and on the order of alkyl chains near the polar headgroup region as observed with 5-doxylstearic acid spin label (5-SASL). The effect of unsaturation is, however, moderate in the membrane center as shown with 16-SASL. Also, the effect of carotenoids on the order and motion of the rigid and highly anisotropic molecules dissolved in the PC membranes is significantly greater in saturated PC membranes.

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847–851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.

3-[ p-(6-Phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PA-DPH): characterization as a fluorescent membrane probe and binding to fatty acid binding proteins

The negatively charged fluorophore 3-[ p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PA-DPH) was characterized by comparison with its parent compound DPH, and with cationic trimethylammonium-DPH (TMA-DPH). The molar absorption coefficient of PA-DPH (60 000 cm −1 · mol −1) as well as its quantum yield (0.7) and fluorescence lifetime (5 ns) in fluid phase membranes are intermediate between DPH and TMA-DPH. Steady-state fluorescence polarization studies show that PA-DPH detects the phase transition of both neutral and anionic bilayers. In fluid phase membranes the absolute values of PA-DPH polarization are considerably higher than DPH and somewhat lower than TMA-DPH. The results suggest that like TMA-DPH, PA-DPH is anchored to the surface of the membrane by its charge, but that it is probing a region somewhat deeper along the bilayer normal. PA-DPH binds to rat hepatic fatty acid binding protein (hFABP) and bovine serum albumin at PA-DPH/protein molar ratios of 1.5:1 and at least 6:1, respectively. Native oleic acid competes with PA-DPH for binding to both proteins, suggesting that the two ligands compete for similar binding sites. The affinity of PA-DPH for hFABP is similar to that of oleic acid. Thus, PA-DPH should be useful both as an anionic fluorescent membrane probe and a long-chain free fatty acid analogue.

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381–410 (1985) Biochim. Biophys. Acta 812, 361–368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.