Cooperativity of the phase transition in single- and multibilayer lipid vesicles.

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.     

The effect of pressure on the phase diagram of mixed dipalmitoyl-dymyristoylphosphatidylcholine bilayers

The application of 136 atm of helium pressure to suspensions of mixed dipalmitoyl-dimyristoylphosphatidylcholine bilayers caused a 3–5°C elevation of points on the envelope of the binary phase diagram.Membrane bilayers containing lateral phase separations are able to respond to external pressure by converting fluid phase phospholipids to the more compact gel phase.     

A spin label study of lipid oxidation catalyzed by heme proteins

Rapid loss of the electron spin resonance signal from a variety of spin labels is observed when ferricytochrome c or metmyogloblin are combined with lipids. Evidence is presented that this loss of signal can be used as a sensitive method to study lipid oxidation catalyzed by heme proteins. Under aerobic conditions and with lipids which bind the heme protein, the kinetics of the oxidation process as observed by the spin label method are identical to the kinetics previously observed by measurements of oxygen uptake. Use of pre-oxidized lipids under anaerobic conditions indicates that cytochrome c reacts with a product of lipid oxidation. Kinetic studies of the anaerobic reaction indicate that cytochrome c reacts rapidly with lipid oxidation products in membrane areas far larger than the area occupied by cytochrome c, implying rapid transport of reactive species within the membrane interior in directions parallel to the membrane surface. Under anaerobic conditions, reaction of cytochrome c with lipid oxidation products appears to produce a relatively long lived (hours) species located in the hydrophobic portion of the membrane, which is capable of subsequent reaction with lipid-soluble spin labels.     

Multiple phase equilibria in binary mixtures of phospholipids

Approximate phase diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data presented here, as well as other earlier spin-label, calorimetric, and X-ray data. These phase diagrams represent an improvement over previous studies in that both solid phases (P_β' and L_β') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two P_β' phases that do not form a continuous range of solid solutions.     

Pressure-induced elevation of phase transition temperature in dipalmitoylphosphatidylcholine bilayers: An electron spin resonance measurement of the enthalpy of phase transition

The application of 136 atm of helium pressure to an aqueous dispersion of dipalmitoylphosphatidylcholine increased the temperature of the primary phase transition at 40.4 ± 0.2 °C by 3.0 °C. The lower temperature pretransition at 30.5 ± 0.5 °C, thought to be due to phosphate headgroup reorganization, was increased by 1.7 °C. Addition of 4% dipalmitoylphosphatidic acid to the dipalmitoylphosphatidylcholine affected the phase transition in the head group region more than in the hydrocarbon chain region. The pressure and temperature data obtained, taken together with the literature value for the bilayer volume expansion during solid-fluid phase transition, and inserted into the Clausius-Clapeyron equation yield a ΔH value of 8.8 kcal/mole for this phase transition. This value is within experimental error of the ΔH value obtained from differential scanning calorimetry and serves to support the validity of the data and the experimental technique. Phase transition was observed by electron spin resonance measurement of the exclusion of the small spin label Tempo (2,2,6,6-tetramethylpiperidine-N-oxyl) from the solid domains of the bilayer. This result offers a possible explanation for the direct antagonism by high pressure of the effects of the inhalation anesthetics.     

Thermal acoustic radiation from multilamellar vesicles in lipid phase transition

A new acoustical method for the investigation of lipid phase transition is introduced based on the measurement of the thermal acoustic radiation (TAR) inherent in lipids. The TAR of multilamellar vesicles from dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) was measured in the megahertz range and the variations in the radiation intensity during the lipid phase transition were recorded. Two types of variations are possible: if the temperature of the vesicles decreases (in the process of transition from the liquid crystalline state to the gel state) then the TAR intensity increases, and if the temperature increases (in the reverse transition) then the TAR intensity decreases. These effects are connected with an increase in the ultrasonic absorption in the vesicles under lipid phase transition. Basing on the results of the TAR investigation, a new theoretical estimate has been developed of the variation in the absorption coefficient during the lipid phase transition. In this estimate, the variation is equated to the ratio of the phase transition entropy to the gas constant.     

The intermediate monoclinic phase of phosphatidylcholines

Two pure phospholipids, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, have been studied using freeze-fracture electron microscopy and the partitioning of the spin label, TEMPO. It is found that the characteristic band pattern, corresponding to monoclinic symmetry in multilamellar liposomes, is observed only in freeze-fracture electron microphotographs when samples are quenched from temperatures intermediate between the chain melting transition temperature and the pretransition temperature of the membrane. Markings are also observed on fracture faces of samples quenched from below the pretransition, but these “bands” are few in number and are widely and irregularly spaced. The lipid membranes used for freeze-fracture were prepared using detergent dialysis and are thought to consist of one, two, or some small number of concentric bilayer shells. These observations are in excellent accord with the recent, prior studies of Janiak, M.J., Small, D.M. and Shipley, G.G., ((1976) Biochemistry, 15, 4575–4580), who found monoclinic symmetry (P_β′ structure) in multimellar liposomes of these phospholipids only when the sample temperature was intermediate between the main, chain melting transition temperature, and the presentation temperature. The significance of these results for relating freeze-fracture electron microphotographis to phase diagrams derived from spin label or calorimetric data is discussed briefly.2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) partitioning data show distinct differences between liposomal preparations of these lipids, and other preparations having fewer bilayers per vesicular structure, with respect to the position, width, and hysteresis of the pretransition.     

Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.     

Spin labels as enzyme substrates Heterogeneous lipid distribution in liver microsomal membranes

Phenobarbital-stimulated microsomal membranes of rabbit liver, containing the cytochrome P450- cytochrome P450 reductase hydroxylating enzyme system in high concentration, have been studied with a version of the spin label technique which uses nitroxide radicals as enzyme substrates. The reduction kinetics of a phosphate ester of tetramethylpiperidine nitroxide (TEMPO-phosphate) and of stearic acid nitroxide by the cytochrome P450 reductase has been studied as a function of the temperature. The Arrhenius plot of the reduction rate constants reveals a striking difference in the behaviour of the water-soluble TEMPO-phosphate label and the lipid-soluble fatty acid label: The activation energy of the fatty acid reduction decreases abruptly at about 32°C from a value of 30.8 kcal/mole to a value of 8.7 kcal/mole, whereas no such break is observed in the Arrhenius plot of the TEMPO-phosphate reduction which yields a value of the activation energy of $\text{ΔW = 13.8}$ kcal/mole in the whole temperature range investigated. Our results clearly indicate the existence of a mosaic-like structure of the membrane with the whole enzyme system being enclosed by a rather rigid phospholipid halo which is in a quasicrystalline structure below 32 °C and undergoes a crystalline-liquid crystalline phase transition at 32 °C, while the bulk lipid of the membrane is in a rather fluid state as reflected by the measured high diffusion coefficient of $\text{D}{}_{\mathrm{<mtext>diff</mtext>}}\text{= 11.0·10}{}^{\mathrm{?8}}\text{cm}{}^2\text{/s}$ at 30 °C and low activation energy of diffusion of $\text{ΔW = 3.85}\text{kcal/mole}$ of a fatty acid spin label incorporated in the membrane.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).     

Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition.

The existence of distinct regions of mismatch in molecular packing at the interfaces of the fluid and ordered domains during the phase transition of dimyristoylphosphatidylcholine vesicles has been demonstrated by measuring the temperature dependence of the permeability to a spin-label cation and comparing this with a statistical mechanical calculation of the fraction of interfacial lipid. The kinetics of uptake and release of the 2,2,6,6-tetramethylpiperidinyl-1-oxycholine (Tempo-choline) spin label by single-bilayer dimyristoylphosphatidylcholine vesicles were measured using electron spin resonance spectroscopy to quantitate the amount of spin label present within the vesicles after removal of the external spin-label by ascorbate at 0 degrees C. Both the uptake and release experiments show that the Tempo-choline permeability peaks to a sharp maximum at the lipid-phase transition, the vesicles being almost impermeable to Tempo-choline below the transition and having a much reduced permeability above. The temperature profile of the permeability is in reasonable quantitative agreement with calculations of the fraction of interfacial boundary lipid from the Zimm and Bragg theory of cooperative transitions, which use independent spin-label measurements of the degree of transition to determine the cooperativity parameter. The relatively high intrinsic permeability of the interfacial regions (P approximately 0.2-1.0 X 10(-8) cm/s) is attributed to the mismatch in molecular packing of the lipid molecules at the ordered-fluid boundaries, which could have important implications not only for permeability in natural membranes (e.g., in transmitter release), but also for the function of membrane-bound enzymes and transport proteins.     

Lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles: A cross-linking study

To determine the nonideal mixing of two lipid components within the membrane, lipid cross-linking experiments were carried out on dipalmitoylphosphatidylethanolamine (DPPE) vesicles and on dipalmitoylphosphatidylethanolamine/dipalmitoylphosphatidylserine (DPPE/DPPS) vesicles. By comparison of the cross-linking reactions on both types of vesicle the mean neighbourhood relations within the binary lipid mixture can be obtained. To elucidate the relationship between cluster formation and phase transition, the temperature dependences of the lipid arrangement within the vesicle membrane and of the lipid order parameter describing the fluidity of the membrane were measured. Cluster size and phase transition correlate: during the phase transition of the lipid species with the lower phase-transition temperature (DPPS) the nonideality of the mixture increases by phase separation. Above the phase transition temperature of the second lipid species (DPPE) the clusters disappear and a slight alternating lipid arrangement is characteristic of the fluid phase.     

Turbidity changes of lipid vesicles near the phase transition temperature as an indication of fusion

Sonicated liposomes of dipalmitoyl phosphatidylcholine show sharp turbidity changes on heating at two distinct temperatures. A decrease in turbidity at the lower temperature (approx. 37°C) is thought to be associated with the phase transition of small vesicles and a decrease at about 44°C with larger vesicles or multilayer. An increase of turbidity between 38 and 43°C is attributed to the fusion of small vesicles. The turbidity changes were studied under various modes of vesicle preparation to confirm the interpretation of the turbidity data. Alternate interpretations are discussed.     

The influence of Ca2+ on the lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles

The lateral lipid distribution within dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylphosphatidylserine (DPPS) vesicle membranes was investigated under the influence of Ca²⁺ using a lipid cross-linking method. To characterize the phase transition in DPPE/DPPS vesicles and to correlate the different phase states of the membrane lipids with the obtained lipid distribution ESR measurements using a fatty acid spin label were carried out. It is shown that Ca²⁺ has a significant influence on the lateral lipid distribution within the fluid phase of the membrane lipids; instead of a slight alternating lipid arrangement in absence of Ca²⁺ due to the electrostatic interaction between the DPPS headgroups after addition of Ca²⁺ a lateral cluster structure is characteristic of the fluid phase.     

Oxidized phospholipids induce phase separation in lipid vesicles

The thermal behaviour of phospholipid multilamellar vesicles (MLV) made of various molar percentages of DPPC and LPPC, containing also oxidized LPPC (LPPCox), was studied by use of EPR spectroscopy and n-DSPC spin label in order to determine variations in the membrane fluidity brought about by lipid oxidation. Experimental variables were temperature, ranging from 4 to 44 degrees C, and molar percentage composition of DPPC/LPPC/LPPCox ternary mixture. We found that the presence of LPPCox in a percentage higher than both normal phospholipids' heavily hindered membrane formation, while lower percentage of the oxidized lipid with higher DPPC percentages yielded two-components EPR spectra, showing the presence of two different fluidity domains, indicative of membrane phase separation. When LPPC was the dominant lipid in the ternary mixture, simple EPR spectra were observed, indicating homogeneity of MLV membranes. Phase separation observed in the presence of LPPCox was better visible at lower temperature (12 degrees C or less), and almost disappeared with increasing temperature (36 degrees C or more). Furthermore, the correlation time of 16-DSPC in ternary mixture MLVs with higher LPPC percentage (homogeneous membranes) was not affected by the presence of LPPCox, while it normally increased upon DPPC percentage increase, as readily calculated from the EPR spectra featuring simple bands at 24 degrees C. It is concluded that oxidized lipid induces phase separation in more rigid DPPC-rich membranes, while leaving fluidity unaffected in more fluid LPPC-rich membranes, and at higher temperature.     

Imipramine and lipid phase transition in inner mitochondrial membrane

As ascertained by freeze-fracture electron microscopy, imipramine prevents lateral phase separation from taking place in inner mitochondrial membranes at sub-zero temperatures. Electron spin resonance (ESR) measurements performed on mitochondrial membranes labeled with the N-oxyl-4′,4′-dimethyloxazolidine derivative of 16-ketostearic acid, show that the spin probe motion is markedly inhibited below 0°C and that 5 mM imipramine attenuates the temperature effect. These results are explained by supposing that imipramine is able to decrease the transition temperature of the inner mitochondrial membrane lipids as it does for simple lipid systems.     

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T(m). In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T(m) and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 microm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.     

Dynamic fluorescence measurements on the main phase transition of dipalmytoylphosphatidylcholine vesicles

The kinetics of the main phase transition in dipalmytoylphosphatidylcholine (DPPC) vesicles have been investigated using our iodine laser-Tjump technique with fluorescence detection. A set of three fluorescent probes has been used to sense different parts of the bilayer hydrocarbon chain region. The well established membrane probes DPH and TMADPH as well as DPHPC, a labelled DPPC molecule. We report three relaxation signals in the s and ms time range, which are detected with all three probes. This result supports our model of the main phase transition in DPPC vesicles.Abbreviations DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmytoylphosphatidylcholine - DPH 1,6-Diphenylhexa-1,3,5-triene - TMADPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene - DPHPC Diphenylhexatriene-phosphatidylcholine - T_m Temperature of the main phase transition     

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.