Time-resolved fluorometric and differential scanning calorimetric investigation of dehydroergosterol in 1-stearoyl-2-caprylphosphatidylcholine bilayers

Thermal and dynamic properties of dehydroergosterol (DHE) in 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine [C(18):C(10)PC] have been studied by differential scanning calorimetry (DSC) and multifrequency phase-modulation fluorometry. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form highly ordered mixed interdigitated bilayers below the maximal transition temperature, Tm, and partially interdigitated bilayers above Tm. This lipid system is thus unique in assessing the interactions between sterols and interdigitated lipid bilayers. DHE is a fluorescent analogue of cholesterol shown in previous studies to behave like cholesterol in noninterdigitated symmetric diacylphosphatidylcholines. DSC data show that DHE exhibits similar characteristics to cholesterol [Chong & Choate (1989) Biophys. J. 55, 551-556] in C(18):C(10)PC bilayers. DHE abolishes the phase transition of C(18):C(10)PC at 27 mol % compared to 25 mol % for cholesterol and decreases Tm, the onset temperature (To), and the completion temperature (Tc), at a similar rate to cholesterol at about -0.25 degrees C per mole percent DHE. Fluorescence data show that the rotational motion of DHE can be described by a hindered anisotropic model. In the gel state of C(18):C(10)PC, the rotational correlation of DHE decreases monotonically with increasing DHE content up to 24 mol %, suggesting that DHE causes a disordering/spacing effect on the packing of mixed interdigitated C(18):C(10)PC bilayers. The rotational correlation time undergoes an abrupt increase from 24 to 27 mol % DHE. Abrupt changes in the DSC parameters were also observed in the neighborhood of 27 mol %, suggesting that major reorganization takes place around this concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential scanning calorimetric study of a homologous series of fully hydrated saturated mixed-chain C(X):C(X + 6) phosphatidylcholines

The successive high-resolution differential scanning calorimetric (DSC) thermograms for aqueous dispersions of a homologous series of mixed-chain phosphatidylcholines, C(X):C(X + 6)PC, have been recorded and analyzed. In this series of saturated mixed-chain phosphatidylcholines, the total number of carbon atoms in the sn-1 acyl chain increases from 11 to 20, and the sn-2 acyl chain is always 6 methylene units longer than the sn-1 acyl chain. In the initial heating DSC thermograms, two prominent endothermic transitions are detected for all the samples prepared from the various C(X):C(X + 6)PCs except C(12):C(18)PC. In contrast, a single exothermic transition is observed on cooling for all the samples except C(13):C(19)PC. The temperature difference between the two endothermic transitions increases linearly as the acyl chain length of C(X):C(X + 6)PC becomes progressively longer. Interestingly, the main phase transition occurs before the subtransition for C(11):C(17)PC dispersions. Our DSC data further demonstrate that the thermodynamic parameters (Tm, delta H, and delta S) associated with the main phase transition for fully hydrated C(13):C(19)PC and other identical MW phosphatidylcholines are inversely related to the corresponding values of the chain-length inequivalence (delta C/CL) for these lipids. This linear relationship can be employed to map the Tm values for aqueous dispersions prepared from a large number of mixed-chain phosphatidylcholines whose values of delta C/CL are within the range of 0.1-0.4.     

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from noninterdigitated gel phases of symmetric diacylphosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cholesterol on the structure and dynamic properties of unsaturated phospholipid bilayers

Molecular dynamics (MD) computer simulations of five different hydrated unsaturated phosphatidylcholine lipid bilayers built up by 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules with 40 mol% cholesterol, and the same five pure phosphatidylcholine bilayers have been performed at 303 K. The simulation box of a lipid bilayer contained 96 phosphatidylcholines, 64 cholesterols, and 3840 water molecules (48 phosphatidylcholine molecules and 32 cholesterols per layer and 24 water molecules per phospholipid or cholesterol in each case). The lateral self-diffusion coefficients of the lipids in these systems and mass density profiles with respect to the bilayer normal have been analyzed. It has been found that the lateral diffusion coefficients of phosphatidylcholine molecules increase with increasing number of double bonds in one of the lipid chains, both in pure bilayers and in bilayers with cholesterol. It has been found as well that the lateral diffusion coefficient of phosphatidylcholine molecules of a lipid bilayer with 40 mol% cholesterol is smaller than that for the corresponding pure phosphatidylcholine bilayer.     

Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes

Self- or concentration quenching of octadecylrhodamine B (C18-Rh) fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the probe concentration is raised to 10 mol%. Cholesterol-dependent enhancement of self-quenching also occurs when N-(lissamine-rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below, as opposed to above, the phase transition. These effects are not due to changes in dimer:monomer absorbance. Stern-Volmer plots indicate a dependence of quenching on nonfluorescent dimers both in the presence and absence of cholesterol. Decreases in fluorescence lifetimes with increasing probe concentration parallel decreases in residual fluorescence of C18-Rh with increasing probe concentration in PC and PC + cholesterol membranes, respectively. Decreases in the steady-state polarization of C18-Rh fluorescence as its concentration is raised to 10 mol% indicate energy transfer with emission between probe molecules in PC and to a lesser extent in PC + cholesterol membranes. The calculated R0 for 50% efficiency of energy transfer from excited state probe to monomer was 55-58 A and to dimer was 27 A. Since lateral diffusion of C18-Rh is probably too slow to permit collisional quenching during the lifetime of the probe, even if C18-Rh were concentrated in a separate phase, C18-Rh self-quenching appears to be due mainly to energy transfer without emission to nonfluorescent dimers.     

Effect of lipid physical state on the rate of peroxidation of liposomes.

The effect of cholesterol on the rate of peroxidation of arachidonic acid and 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) in dimyristoylphosphatidylcholine (DMPC) liposomes was examined above and below the phase transition temperature (Tm) of the lipid. The rate of peroxidation of arachidonic acid was more rapid below the phase transition temperature of the host lipid. At a temperature below the Tm (4 degrees C), increasing concentrations of cholesterol reduced the rate of peroxidation of arachidonic acid as judged by the production of thiobarbituric acid reactive substances. Above Tm (37 degrees C), cholesterol increased the rate of peroxidation of the fatty acid. Similarly, PAPC was peroxidized more rapidly at 4 degrees C than at 37 degrees C. However, cholesterol had little effect on the rate of peroxidation of PAPC at 4 degrees C. The rate of peroxidation of arachidonic acid was related to the lipid bilayer fluidity as judged by fluorescence anisotropy measurements of diphenylhexatriene. The rate of peroxidation increased slowly with increasing rigidity of the probe environment when the bilayer was relatively fluid and more rapidly as the environment became more rigid. The increase in the rate of peroxidation of arachidonic acid in the less fluid host lipid was unrelated to differences in iron binding or to transfer of arachidonic acid to the aqueous phase. Decreasing the concentration of arachidonic acid in DMPC to less than 2 mol% dramatically decreased the rate of peroxidation at 4 degrees C, suggesting that formation of clusters of fatty acids at 4 degrees C is required for rapid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The bilayer melting transition in lung surfactant bilayers: the role of cholesterol

Aqueous dispersions of a porcine lung surfactant (PLS) extract with and without cholesterol supplementation were analyzed by X-ray scattering. Lamellar liquid-crystalline and gel-type bilayer phases are formed, as in pure phosphatidylcholine (PC)-cholesterol systems. This PLS extract, developed for clinical applications, has a cholesterol content of less than 1% (w/w). Above the limit of swelling, the bilayer structure shows a melting (main) transition during heating at about 34 degrees C. When 13 mol% cholesterol was added to PLS, so that the cholesterol content of natural lung surfactant was reached, the X-ray scattering pattern showed pronounced changes. The main transition temperature was reduced to the range 20-25 degrees C, whereas according to earlier studies of disaturated PC-cholesterol bilayers in water the main transition remains almost constant when the amount of solubilized cholesterol is increased. Furthermore, the changes in scattering pattern at passing this transition in PLS-cholesterol samples were much smaller than at the same transition in PLS samples. These effects of cholesterol solubilization can be related to phase segregation within the bilayers, known from pure PC-cholesterol systems. One phase, solubilizing about 8 mol% cholesterol, exhibits a melting transition, whereas the other bilayer phase, with a liquid-crystalline disordered conformation, has a cholesterol content in the range 20-30 mol% and this phase shows no thermal transition. The relative amount of bilayer lipids that is transformed at the main transition in the PLS-cholesterol sample is therefore only half compared to that in PLS samples. The reduction in transition temperature in the segregated bilayer of lung surfactant lipids is probably an effect of enrichment of disaturated PC species in the phase, which is poor in cholesterol. This work indicates that cholesterol in lung surfactant regulates the crystallization behavior.     

Effects of different cultivation temperatures on plasma membrane ATPase activity and lipid composition of sugar beet roots.

Sugar beet seedlings (Beta vulgaris L. cv. Monohill) were cultivated for 3 weeks at different root and shoot temperatures and the plasma membranes (PM) from roots were purified by aqueous two-phase partitioning and analyzed for lipid composition and ATPase activities. Lipid analyses, undertaken immediately after PM purification from the roots, showed that a low root zone temperature (10 degrees C) decreased the ratio between the major lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A low temperature in the root environment increased the mol% of PE and decreased the mol% of phosphatidic acid (PA), independent on the shoot growth temperature. A low temperature also decreased the mol% of linoleic acid (18:2) and increased mol% of linolenic acid (18:3) in the analyzed lipid classes, especially in PC and PE. The ratio between acyl chain lipids and protein generally increased in PM from roots grown at 10 degrees C, compared with higher temperature. The changes in lipid composition correlated with changes in ATPase activities, detected as hydrolyses of MgATP. The kinetic parameters, K(m) and V of the PM H(+)ATPase in roots increased at a low cultivation temperature, independent on shoot temperature. Moreover, Arrhenius analyses showed that the transition temperature was independent of both root or shoot growth temperature at 10-24 degrees C, whereas the activation energy of the ATPase was dependent on the growth temperature of the root, and independent on shoot temperature. Thus, acclimation processes can take place in roots, irrespective of the shoot temperature.     

Scanning calorimetric study of fully hydrated asymmetric phosphatidylcholines with one acyl chain twice as long as the other

The asymmetric C(18):C(10)PC molecules are known by X-ray diffraction to self-assemble, in excess water, into a lamellar structure known as the mixed interdigitated bilayer at T less than Tm. In this structure, the long C(18)-acyl chain is interdigitated fully across the entire hydrocarbon width of the bilayer, while the shorter C(10)-acyl chain, which is about half as long as the C(18)-acyl chain, packs end to end with a C(10)-acyl chain of another lipid molecule in the opposing bilayer leaflet. We have synthesized the following asymmetric phosphatidylcholines (PC's): C(16):C(9)PC, C(16):C(10)PC, C(18):C(10)PC, C(18):C(11)PC, C(20):C(11)PC, C(20):C(12)PC, C(22):C(12)PC, C(22):C(13)PC, C(8):C(18)PC, and C(10):C(22)PC. These 10 asymmetric phosphatidylcholines have a common characteristic; i.e., the length of the longer extended acyl chain is about twice as long as that of the shorter acyl chain. On the basis of the known lamellar structure of C(18):C(10)PC, we anticipate that these asymmetric phosphatidylcholines will also form mixed interdigitated bilayers. We have employed high-resolution differential scanning calorimetry (DSC) to investigate the thermotropic behavior of liposomes prepared from these asymmetric phosphatidylcholines. If our anticipation is correct, one would find that the thermodynamic data (Tm, delta H, or delta S) associated with the main thermal phase transitions of these asymmetric phosphatidylcholine dispersions will fit into a continuous curve as they are plotted as a function of the hydrocarbon width of the putative mixed interdigitated bilayer. Experimental data presented in this paper indeed bear this out. For comparison, a DSC study of multilamellar dispersions prepared from a series of saturated symmetric phosphatidylcholines has also been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal history alters cholesterol effect on transition of 1-palmitoyl-2-linoleoyl phosphatidylcholine.

The effect of cholesterol on the bilayer phase behavior of heteroacid phosphatidylcholines with one unsaturated fatty acid depends on the nature of the unsaturated chain. Previous differential scanning calorimetry (DSC) studies showed that 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC) had a broad, weak transition at about -18 degrees C, which was effectively eliminated by less than 15 mol% cholesterol. Phospholipids with greater and lesser degrees of unsaturation displayed stronger phase transitions and less sensitivity to cholesterol. In this work, deuterium nuclear magnetic resonance has been used to examine the phase behavior of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC-d31) alone, and with 15 mol % cholesterol. The behavior is found to be sensitive to sample thermal history. Moderately fast cooling (1 degree/h) results in a continuous phase change from a fluid to an ordered phase in the pure lipid. Under similar cooling conditions, the sample containing cholesterol displays increased chain order and a continuous phase change with no apparent isothermal transition. However, when these systems are cooled at a reduced rate (0.3 degree/h), the continuous phase change is pre-empted by a sharp transition into a more ordered phase that gives a deuterium spectrum having intensity at a value of the quadrupole-splitting characteristic of a rigid lattice system. In the pure lipid, this transition effectively coincides with the center of the continuous phase change. Addition of 15 mol % cholesterol lowers the temperature of this sharp transition by about 3 degrees C. These observations provide some insights into the behavior of this system seen using differential scanning calorimetry. Results of deuteron transverse relaxation measurements under these conditions are also reported.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Cholesterol-multilipid interactions in bilayers

To extend our knowledge of model membrane systems based upon one lipid component, multi-lamellar bilayers were made of cholesterol with two phospholipids in equimolar ratio, and the enthalpy change delta H of the main phase transition of the temary mixture was measured by differential scanning calorimetry (DSC) as a function of increasing cholesterol concentration c. The lipids were saturated phosphatidylcholines CnPC of acyl chain length n, and as the n of the two lipids became more different (from C13PC/C14PC to C14PC/C15PC to C14PC/C18PC to C14PC/C19PC) distinct breaks in the delta H versus c plots were observed. These mixtures displayed only one broad DSC endotherm. Mixtures of an unsaturated lipid C18: 1PC (dioleoyl) with C16PC or with C18PC showed two peaks, with each peak being associated with its parent lipid. However, the delta H versus c plots for each of these peaks showed an initial independence of cholesterol concentration followed by a dependence on cholesterol concentration. These results indicate that, in lipid mixtures, the type of interaction of cholesterol with each lipid component depends on the concentration of cholesterol present.     

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.     

Circular dichroism study of membrane dynamics focused on effect of monosialogangliosides

Monosialogangliosides (GM) purified from bovine brain were incorporated into circular dichroism (CD)-active liposomes and the effects of GM on the membrane dynamics were studied by CD spectroscopy. In the presence of 7 mol% of GM, the phase transition temperature (Tc) of the membrane increased by ca. 10 degrees C compared with the membrane without GM and characteristic CD spectra were observed for CD-active liposomes incorporating GM at low temperature. Asialogangliosides had no effect on the CD spectra or Tc. We have also studied the role of GM in reducing leakage of [3H]sucrose from liposomes composed of egg phosphatidylcholine, dipalmitoyl phosphatidic acid, cholesterol and alpha-tocopherol with a molar ratio of 4 : 1 : 5 : 0.1 in the presence of human plasma at 25 degrees C. The half-life of [3H]-sucrose leakage was 173 h for liposomes incorporating 7 mol% of GM. On the other hand, the half-lives for liposomes incorporating 7 mol% of asialogangliosides and liposomes without glycolipids were 45 and 42 h, respectively. These results indicate that sialic acid on the membrane surface contributes to the increase of Tc, to the change of the aggregation state of phospholipids and to the stabilization of liposomes in plasma.     

Effect of ganglioside-GM1 on the order of phosphatidylcholine-cholesterol multilamellar liposomes. A fluorescence polarization study

The effect(s) of bovine brain ganglioside-GM1 on the order of phosphatidylcholine-cholesterol membranes were studied using steady-state fluorescence polarization (FPZ) techniques with 1,6-diphenyl-1,3,5-hexatriene (DPH) as the membrane probe. In the absence of cholesterol, GM1 (30 mol%) increases both membrane order and the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) membranes. However, in the presence of cholesterol (0.3 or 0.5, cholesterol/phospholipid molar ratio), GM1 significantly decreases steady-state anisotropy (rs) at temperatures above the Tm for the particular phospholipid. This effect may, in part relate to a dilution of membrane cholesterol and is shared by bovine brain sphingomyelin (SM). GM1 (30 mol%) increases the order of 1-palmityl-2-oleyl-PC (POPC) membranes. However, in the presence of cholesterol (0.3 molar ratio) GM1 neither increases or decreases order. Thus, in cholesterol containing artificial membranes, the effect of GM1 depends on the phosphatidylcholine (PC) fatty acid composition and may not be evident from the effect of GM1 on pure PC membranes.     

Molecular mechanics and calorimetric studies of phosphatidylethanols

Phosphatidylethanols (PEths) are negatively charged diacyl phospholipids that are ubiquitously present in humans under the condition of alcohol intoxication. These lipids, derived in vivo from other naturally occurring phospholipids such as phosphatidylcholines (PC) via transphosphatidylation reaction as catalyzed by phospholipase D in the presence of ethanol, are well known to affect many biochemical properties of the cell membranes in humans. In this communication, we applied the combined approach of molecular mechanics (MM) simulations and high-sensitivity differential scanning calorimetry (DSC) to investigate the structure and phase transition behavior of PEth. We first determined the energy-minimized structures of tetrameric C(15):C(15)PEth arranged in two types of packing motif by the MM approach. An inwardly bent orientation of the lipid headgroup was observed; specifically, the methyl terminus of PEth's headgroup was juxtaposed intramolecularly to the C(2) atom of the sn-2 acyl chain. Clearly, this headgroup conformation was rather unique among all naturally occurring phospholipids. Subsequently, the phase transition behavior of the fully hydrated lipid bilayers prepared individually from 11 species of saturated C(X):C(Y)PEth with the same MW was studied by DSC, and the resulting Tm values were codified in terms of the normalized acyl chain asymmetry (deltaC/CL). A V-shaped Tm profile was observed in the plot of Tm versus deltaC/CL for each subclass of these lipids, suggesting two types of packing motif for C(X):C(Y)PEth at T < Tm. Moreover, it was observed that within each packing motif these Tm values were, on average, 2.0 +/- 0.9 degrees C smaller than the Tm values of the corresponding saturated PC. However, based on the unique headgroup conformation of PEth, we were able to predict that monounsaturated PEth with a cis double bond near the H2O/hydrocarbon interface would exhibit a higher Tm than the corresponding PC. Most interestingly, this prediction was indeed borne out by DSC results obtained with C(18):C(20:1delta5)PC and C(18):C(20:1delta5)PEth.     

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

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

A thermodynamic study of the effects of cholesterol on the interaction between liposomes and ethanol

The association of ethanol with unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes of varying cholesterol content has been investigated by isothermal titration calorimetry over a wide temperature range (8-45 degrees C). The calorimetric data show that the interaction of ethanol with the lipid membranes is endothermic and strongly dependent on the phase behavior of the mixed lipid bilayer, specifically whether the lipid bilayer is in the solid ordered (so), liquid disordered (ld), or liquid ordered (lo) phase. In the low concentration regime (<10 mol%), cholesterol enhances the affinity of ethanol for the lipid bilayer compared to pure DMPC bilayers, whereas higher levels of cholesterol (>10 mol%) reduce affinity of ethanol for the lipid bilayer. Moreover, the experimental data reveal that the affinity of ethanol for the DMPC bilayers containing small amounts of cholesterol is enhanced in the region around the main phase transition. The results suggest the existence of a close relationship between the physical structure of the lipid bilayer and the association of ethanol with the bilayer. In particular, the existence of dynamically coexisting domains of gel and fluid lipids in the transition temperature region may play an important role for association of ethanol with the lipid bilayers. Finally, the relation between cholesterol content and the affinity of ethanol for the lipid bilayer provides some support for the in vivo observation that cholesterol acts as a natural antagonist against alcohol intoxication.     

Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration

We have measured the lateral diffusion coefficient (D), of active dansyl-labeled gramicidin C (DGC), using the technique of fluorescence photobleaching recovery, under conditions in which the cylindrical dimer channel of DGC predominates. In pure, hydrated, dimyristoylphosphatidylcholine (DMPC) multibilayers (MBL), D decreases from 6 X 10(-8) cm2/s at 40 degrees C to 3 X 10(-8) cm2/s at 25 degrees C, and drops 100-fold at 23 degrees C, the phase transition temperature (Tm) of DMPC. Above Tm, addition of cholesterol decreases D; a threefold stepwise drop occurs between 10 and 20 mol %. Below Tm, increasing cholesterol increases D; a 10-fold increase occurs between 10 and 20 mol % at 21 degrees C, between 20 and 25 mol % at 15 degrees C, and between 25 and 30 mol % at 5 degrees C. In egg phosphatidylcholine (EPC) MBL, D decreases linearly from 5 X 10(-8) cm2/s at 35 degrees C to 2 X 10(-8) cm2/s at 5 degrees C; addition of equimolar cholesterol reduces D by a factor of 2. Thus this transmembrane polypeptide at low membrane concentrations diffuses quite like a lipid molecule. Its diffusivity in lipid mixtures appears to reflect predicted changes of lateral composition. Increasing gramicidin C (GC) in DMPC/GC MBL broadened the phase transition, and the diffusion coefficient of the lipid probe N-4-nitrobenzo-2-diazole phosphatidylethanolamine (NBD-PE) at 30 degrees C decreases from 8 X 10(-8) cm2/s below 5 mol % GC to 2 X 10(-8) cm2/s at 14 mol % GC; D for DGC similarly decreases from 4 X 10(-8) cm2/s at 2 mol % GC to 1.4 X 10(-8) cm2/s at 14 mol % GC. Hence, above Tm, high concentrations of this polypeptide restrict the lateral mobility of membrane components.     

Modification by diacylglycerol of the structure and interaction of various phospholipid bilayer membranes

The effects of incorporating diacylglycerol (DG) derived from egg phosphatidylcholine (PC) into PC, egg phosphatidylethanolamine (PE), and bovine phosphatidylserine (PS) have been measured. In excess solution DG induces a multilamellar-to-hexagonal (L-H) structural transition in PE and PC that is temperature dependent. At 37 degrees C it begins at about 3 and 30 mol%, respectively. In PC at lower DG concentrations a modified lamellar phase is formed; at about 70 mol% DG a single primitive cubic phase forms. An L-H transition induced by 20-30 mol% DG in PS is dependent on ionic strength and degree of lipid hydration, with the appearance of crystalline acyl chains at the higher DG levels. Calcium precipitates of DG/PS (1/1) mixtures have melted chains. Structural parameters were derived for the lamellar phases at subtransition levels of DG in PE and PC. The area per polar group is increased, but by contrast with cholesterol, the polar group spreading is not accompanied by an increase in bilayer thickness. DG does not affect the equilibrium separation of PC or PE bilayers. Measured interbilayer forces as they vary with bilayer separation show that DG at 20 mol% does not effect closer apposition of PC bilayers at any separation. Spreading the polar groups may effect the binding of protein kinase C or the activation of phospholipases; the nonlamellar phases may be linked to the biochemical production of DG in cellular processes involving membrane fusion.