Vesicles from mixtures of bipolar archaebacterial lipids with egg phosphatidylcholine

Bipolar lipids from the membranes of archaebacterium Caldariella acidophila can form small unilamellar liposomes, when sonicated from lipid mixtures containing at least 25 mol% egg phosphatidylcholine. With increasing contents of archaebacterial lipid the inner radius of highly sonicated vesicles increases (from approx. 90 Å to approx. 160 Å) concomitant with an enhanced asymmetric distribution of the phosphatidylcholine molecules towards the outer face of the lipid bilayer membranes.     

Influence of phosphatidylcholine/cholesterol molar ratios in liposomes on cholesterol reactivity with cholesterol:oxygen oxidoreductase]

The reactivity of sonicated phosphatidylcholine-cholesterol liposomes with cholesterol : oxygene oxydoreductase, an enzyme which catalyses the oxidation of the 3 beta hydroxyl group of cholesterol to a ketone group, is compared with that of ternary system phosphatidylcholine-cholesterol-Thesit. Regardless to the phosphatidylcholines nature and the phosphatidylcholine/cholesterol molar ratio (R), the enzymatic oxidation rate of liposomal cholesterol is slower than when the reaction is developed in the present of Thesit, a surfactif agent which destroyes the lamellar particles. This is true whether Thesit is added during preparation of dispersions or during incubation with cholesterol oxydase. The enzymatic oxydation rate of cholesterol of ternary systems phosphatidylcholine-cholesterol-Thesit is independent of the (R) value and the phosphatidylcholine fatty acid unsaturation, whereas that of phosphatidylcholine-cholesterol dispersions depends on these two parameters. The reaction rate increases in the order: dipalmitoylphosphatidylcholine to yolk egg phosphatidylcholines, and dioleylphosphatidylcholine. The optimal conditions for cholesterol oxidation were found to be R = 0.5. This result is not affected by the phosphatidylcholines nature. In order to explain these data, various hypotheses are considered. In particular, the weak liposomal cholesterol reactivity with cholesterol oxidase could result from an inhibitory effect on the enzyme-substrate combination due to the polar phosphorylcholine groups.     

Interaction of differently oriented lipids in monolayer: mixed monolayers of 16-(9-anthroyloxy)palmitic acid with phosphatidylcholine and cholesterol

16-(9-Anthroyloxy)palmitic acid (16-AP) is a bifunctional molecule with carboxyl and 9-anthroyloxy groups attached at both ends of the hydrocarbon chain. At the air-water interface, in a monolayer, the 16-AP molecule has horizontal and vertical orientations, depending on the surface pressure of the monolayer. The miscibilities of 16-AP with dimyristoylphosphatidylcholine (DMPC), cholesterol (CH), and fatty acids in mixed monolayers were evaluated in investigations of monolayer phase transitions. Lipid molecules with flexible hydrocarbon chains, i.e., DMPC and fatty acids, formed homogeneous mixed monolayers with horizontally oriented 16-AP. On the other hand, the rigid molecule, CH, could not accommodate the horizontally oriented 16-AP in a monolayer, and there was a phase separation from 16-AP. In biological and reconstituted membranes, preferential binding of phospholipid to the integral protein and exclusion of cholesterol in close vicinity of the membrane protein have been recognized. On the basis of this work, it can be expected that flexible lipids readily accommodate the rough hydrophobic surface of integral proteins and stabilize the structure of the protein, while rigid lipids such as cholesterol are removed from the immediate environment of the membrane protein, if the protein does not interact specifically with the rigid lipids.     

Extraction of cholesterol from biological membranes with positively charged micelles of phosphatidylcholine

The content of cholesterol in red cell and platelet membranes was lowered in rabbits with experimental atherosclerosis after intravenous injection of positively charged micelles of soybean phosphatidylcholine. That lowering was accompanied by a reduction in membrane microviscosity, rise of the activity of Na,K- and Ca-ATPases of red cells, and a decrease in the rate of the ADP- and collagen-induced platelet aggregation. Injection of phosphatidylcholine gave rise to an increase in the blood serum content of phospholipids and cholesterol in high density lipoprotein fractions, to a reduction in the content of triglycerides and the atherogenicity index, as well as to the lowering of the microviscosity of high density lipoproteins. The aortal area affected by atherosclerotic lesions was 2 times less in the group of animals given phosphatidylcholine.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Some properties of particles of egg-yolk phosphatidylcholine and cholesterol

Some physicochemical properties of particles of phosphatidylcholine and cholesterol, produced by ultrasonication of their aqueous dispersions in varying proportions and concentrations were investigated. Cholesterol altered the micellar characteristics according to its proportion. S_{20, w} values increased incrementally from 2.53 for phosphatidylcholine alone to 15.65 for particles containing the highest proportion and concentration of solubilized cholesterol (0.28 g/100 ml of each lipid). Similarly, particle weight increased from 3 × 10⁶ to 7.12 × 10⁷, axial ratios from 3.9 (discs) and 3.3 (rods) to 28.0 (discs) and 13.0 (rods), the viscosity shape factor, ν, from 3.9 to 20.7, water of hydration decreased from 0.26 g H₂O/g lipid to 0.22 g H₂O/g lipid and the number of monomers within each particle ranged from 4000 to 84,000.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Interactions of proteins and cholesterol with lipids in bilayer membranes

Mixtures of lipids and proteins, 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 (TEMPO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$, of the lipid and aggregated below $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$. For mixtures af dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains was 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 temperarture of the lipid is discussed.     

The affinity of cholesterol for phosphatidylcholine and sphingomyelin.

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for N-palmitoyl sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.     

Peroxidation of phosphatidylcholine and cholesterol in mixed monolayers]

Peroxidation of phosphatidylcholine and cholesterol in mixed monolayers at the air-water surface is studied. It is shown that the rate of phosphatidylcholine peroxidation is abruptly decreased in the presence of cholesterol.     

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.     

Phase equilibria in binary mixtures of phosphatidylcholine and cholesterol

The paramagnetic resonance spectra of two spin-labels, 2,2,6,6-tetramethylpiperadinyl-1-oxy and a head-group spin-labeled phosphatidylethanolamine (L-alpha-dipalmitoylphosphatidyl-N-ethanolamine), have been used to study solid-liquid and liquid-liquid phase separations in binary mixtures of dimyristoylphosphatidylcholine and cholesterol. A quantitative analysis of these resonance spectra supports the view that at temperatures below theta m, the chain-melting temperature of the phospholipid, and at cholesterol mole fractions Xc less than 0.2, these mixtures consist of two phases, a solid phase of essentially pure dimyristoylphosphatidylcholine and a fluid phase having a mole fraction of cholesterol equal to 0.2. The spin-label data also provide evidence for fluid-fluid immiscibility in the bilayer membrane at temperatures above the chain melting transition temperature of dimyristoylphosphatidylcholine.     

The effect of cholesterol on the structure of phosphatidylcholine bilayers

The effect of cholesterol on the structure of phosphatidylcholine bilayers was investigated by X-ray diffraction methods. Electron density profiles at 5 Å resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayer was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12–16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.     

EPR investigation of clomipramine interaction with phosphatidylcholine membranes in presence and absence of cholesterol

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.     

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.     

Calcium association with phosphatidylserine Modification by cholesterol and phosphatidylcholine in monolayers and bilayers

We have examined the association of Ca²⁺ with phosphatidylserine/cholesterol and phosphatidylserine/ dimyristoylphosphatidylcholine mixed monolayers using a surface radiocounting technique. No Ca²⁺ association with pure monolayers of the uncharged molecules was observed. The Ca²⁺/phosphatidylserine surface ratio was approximately 1:2 in expanded monolayers of the pure anionic lipid and in phosphatidylserine/phosphatidylcholine mixtures. An increase in surface-associated Ca²⁺ to a number ratio of 1:1 was observed in phosphatidylserine/cholesterol films when the mole fraction of cholesterol was raised to 0.5 and above and the phospholipid number density held constant. We interpret these findings as a prevention of intermolecular salt formation by the sterol. Further support is provided by particle electrophoresis     

Reaction of lecithin:cholesterol acyltransferase with micellar complexes of apolipoprotein A-I and phosphatidylcholine, containing variable amounts of cholesterol

In a continued investigation of lecithin:cholesterol acyltransferase reaction with micellar, discoidal complexes of phosphatidylcholine (PC) . cholesterol . apolipoprotein A-I (apo-A-I), we prepared well defined complexes with variable free cholesterol contents and examined their reactivity with purified enzyme. The complexes, prepared by the sodium cholate dialysis method, were fractionated into "small" and "large" classes by gel filtration of the reaction mixtures through a Bio-Gel A-5m column. The small complexes had egg-PC/cholesterol/apo-A-I molar ratios from 68:14:1 to 80:1:1, discoidal shapes with diameters around 114 (+/- 13) A and widths of 42 A by electron microscopy, and Stokes radii from 47 to 49 A corresponding to molecular weights near 2 X 10(5). The corresponding properties of the large complexes, isolated from samples with higher cholesterol contents, were egg-PC/cholesterol/apo-A-I molar ratios from 84:26:1 to 96:17:1, diameters of 161 (+/- 20) A, widths of 43 A, Stokes radii around 80 A, and estimated molecular weights in the vicinity of 5 X 10(5). Both types of complexes, when adjusted to equal apo-A-I concentrations, gave essentially identical initial reaction velocities with purified lecithin:cholesterol acyltransferase over a wide range of cholesterol concentrations (from 2 X 10(-7) to 4 X 10(-4) M), PC/cholesterol molar ratios (from 3:1 to 12:1), and quite different lipid fluidity conditions as detected by diphenylhexatriene fluorescence polarization. When complexes were adjusted to a constant cholesterol concentration, the initial velocities of the lecithin:cholesterol acyltransferase reaction followed Michaelis-Menten kinetics relative to the apo-A-I concentrations. Arrhenius plots of initial reaction rates for various complexes with variable cholesterol content and fluidity, measured at constant apo-A-I concentrations, gave identical temperature dependences with an average activation energy of 18.0 kcal/mol. These results strongly suggest that the cholesterol esterification on high density lipoprotein particles does not depend on their unesterified-cholesterol contents, PC/unesterified-cholesterol molar ratios, nor on the fluidity of their lipid domains.     

Effect of cholesterol on the ethanol-induced interdigitated gel phase in phosphatidylcholine: use of fluorophore pyrene-labeled phosphatidylcholine

It is now recognized that many amphiphilic molecules such as ethanol can induce the formation of the fully interdigitated gel phase (L beta I) in phosphatidylcholines (PC's). In the present study, we have developed a simple detection method for the L beta I phase using pyrene-labeled PC (PyrPC), which is a PC analogue with covalently coupled pyrene moiety at the end of one of its acyl chains. The intensity ratio of its fluorescence vibrational bands is a reflection of the polarity of the environment of the fluorophore. We have tested this fluorophore in several established interdigitated lipid systems, including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC) in the presence of high concentrations of ethanol and 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC) and 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC) in the absence of any additives. We have found in each of these systems that the ratio of the intensities of band III (387.5 nm) to band I (376.5 nm) is sensitive to the lipid phase change from the noninterdigitated L beta' phase to the interdigitated L beta I phase. By comparison of the III/I ratios for PyrPC in the lipid systems with the III/I ratios for methylpyrene in organic solvents, it was shown that the polarity of the PyrPC environment in the L beta I phase is similar to that of pentanol or ethanol. Using this method, we investigated the effect of cholesterol on the ethanol induction of the interdigitated gel phase in 1,2-DPPC. We found that the ethanol induction of the interdigitated gel phase is prevented by the presence of 20 mol % cholesterol.     

Characterization of antibody to human phosphatidylcholine: cholesterol acyltransferase.

Purified preparations of phosphatidylcholine (lecithin): cholesterol acyltransferase (EC 2.3.1.43), were injected into goats to produce antisera reacting with this enzyme. The antisera and the gamma-globulin derived thereform were examined by the technics of immunodiffusion, immunoelectrophoresis and immunoinhibition of the enzyme. The antisera gave no precipitation lines with human high density lipoproteins (HDL) and human low density lipoproteins (LDL). A weak antibody titer towards human serum albumin was noted only after prolonged immunization. The enzymatically active band isolated from acrylamide gels gave a single arc in immunodiffusion and immunoelectrophoresis. The gamma-globulin derived from the antisera inhibited human phosphatidylcholine:cholesterol acyltransferase activity.