Influence of the lamellar phase unbinding energy on the relative stability of lamellar and inverted cubic phases.

Based on curvature energy considerations, nonbilayer phase-forming phospholipids in excess water should form stable bicontinuous inverted cubic (Q(II)) phases at temperatures between the lamellar (L(alpha)) and inverted hexagonal (H(II)) phase regions. However, the phosphatidylethanolamines (PEs), which are a common class of biomembrane phospholipids, typically display direct L(alpha)/H(II) phase transitions and may form intermediate Q(II) phases only after the temperature is cycled repeatedly across the L(alpha)/H(II) phase transition temperature, T(H), or when the H(II) phases are cooled from T > T(H). This raises the question of whether models of inverted phase stability, which are based on curvature energy alone, accurately predict the relative free energy of these phases. Here we demonstrate the important role of a noncurvature energy contribution, the unbinding energy of the L(alpha) phase bilayers, g(u), that serves to stabilize the L(alpha) phase relative to the nonlamellar phases. The planar L(alpha) phase bilayers must separate for a Q(II) phase to form and it turns out that the work of their unbinding can be larger than the curvature energy reduction on formation of Q(II) phase from L(alpha) at temperatures near the L(alpha)/Q(II) transition temperature (T(Q)). Using g(u) and elastic constant values typical of unsaturated PEs, we show that g(u) is sufficient to make T(Q) > T(H) for the latter lipids. Such systems would display direct L(alpha) --> H(II) transitions, and a Q(II) phase might only form as a metastable phase upon cooling of the H(II) phase. The g(u) values for methylated PEs and PE/phosphatidylcholine mixtures are significantly smaller than those for PEs and increase T(Q) by only a few degrees, consistent with observations of these systems. This influence of g(u) also rationalizes the effect of some aqueous solutes to increase the rate of Q(II) formation during temperature cycling of lipid dispersions. Finally, the results are relevant to protocols for determining the Gaussian curvature modulus, which substantially affects the energy of intermediates in membrane fusion and fission. Recently, two such methods were proposed based on measuring T(Q) and on measuring Q(II) phase unit cell dimensions, respectively. In view of the effect of g(u) on T(Q) that we describe here, the latter method, which does not depend on the value of g(u), is preferable.

Thermal stability of calf skin collagen type I in salt solutions.

The thermal stability of acid-soluble collagen type I from calf skin in salt solutions is studied by high-sensitivity differential scanning calorimetry. Three concentration ranges have been clearly distinguished in the dependence of collagen thermal stability on ion concentration. At concentrations below 20 mM, all studied salts reduce the temperature of collagen denaturation with a factor of about 0.2 degree C per 1 mM. This effect is attributed to screening of electrostatic interactions leading to collagen stabilisation. At higher concentrations, roughly in the range 20-500 mM, the different salts either slightly stabilise or further destabilise the collagen molecule in salt-specific way that correlates with their position in the lyotropic series. The effect of anions is dominating and follows the order H2PO4- > or = SO4(2-) > Cl- > SCN-, with sign inversion at about SO4(2-). This effect, generally known as the Hofmeister effect, is associated with indirect protein-salt interactions exerted via competition for water molecules between ions and the protein surface. At still higher salt concentrations (onset concentrations between 200 and 800 mM for the different salts), the temperature of collagen denaturation and solution opacity markedly increase for all studied salts due to protein salting out and aggregation. The ability of salts to salt out collagen also correlates with their position in the lyotropic series and increases for chaotropic ions. The SO4(2-) anions interact specifically with collagen - they induce splitting of the protein denaturation peak into two components in the range 100-150 mM Na2SO4 and 300-750 mM Li2SO4. The variations of the collagen denaturation enthalpy at low and intermediate salt concentrations are consistent with a weak linear increase of the enthalpy with denaturation temperature. Its derivative, d(delta H)/dT, is approximately equal to the independently measured difference in the heat capacities of the denatured and native states, delta Cp = Cp(D) - Cp(N) approximately 0.1 cal.g-1 K-1.

Rapid reversible formation of a metastable subgel phase in saturated diacylphosphatidylcholines.

Formation of well ordered lamellar subgel (SGII) phase in aqueous dispersions of L-dipalmitoylphosphatidylcholine upon cooling from the lamellar gel phase, without low-temperature equilibration, is observed in real time using synchrotron x-ray diffraction. It has the same lamellar repeat period as the gel phase from which it was formed but differs in its wide-angle diffraction pattern. The SGII phase forms at about 7 degrees C upon cooling at 2 degrees C/min. In temperature jump experiments at 1 degree C/s from 50 to -5 degrees C, the relaxation time of the lamellar gel-SGII transition is found to be approximately 15 s. The conversion between the lamellar gel and SGII phase is cooperative and rapidly reversible. Upon heating, it coincides in temperature with an endothermic event with a calorimetric enthalpy of 0.35 kcal/mol, the so-called sub-subtransition. Similar sub-subtransitions are also observed calorimetrically at temperatures approximately 10 degrees C below the subtransition, without low-temperature storage, in aqueous dispersions of L-dimyristoylphosphatidylcholine and L-distearoylphosphatidylcholine, but not in racemic DL-dipalmitoylphosphatidylcholine. The formation of the equilibrium lamellar crystalline Lc phase appears to take place only from within the SGII phase.

Different phase behaviour of the sn-1 and sn-3 stereoisomers of the glycolipid di-tetradecyl-beta-D-galactosylglycerol.

The phase behaviour of a synthetic, stereochemically pure glycolipid 2,3-di-O-tetradecyl-1-O-beta-D-galactosyl-sn-glycerol (14-2,3-Gal) in excess water has been characterized by differential scanning calorimetry and time-resolved X-ray diffraction and compared with that of the previously studied sn-3 stereoisomer, 1,2-di-O-tetradecyl-3-O-beta-D-galactosyl-sn-glycerol (14-1,2-Gal), and 1,2-di-O-tetradecyl-3-O-beta-D-glucosyl-sn-glycerol (14-1,2-Glc). The properties of 14-1,2-Gal and 14-2,3-Gal are completely different with respect to phase sequences, metastable behaviour, transition temperatures and enthalpies, but there is a rather close similarity between the phase patterns of 14-2,3-Gal and 14-1,2-Glc. The sn-3 stereoisomer, 14-1,2-Gal, exhibits a direct lamellar crystalline to inverted hexagonal phase transition (Lc-->HII) on heating and a HII-->L alpha (metastable)-->L beta(metastable)-->Lc phase sequence in subsequent cooling, while both 14-2,3-Gal and 14-1,2-Glc are characterized by an Lc-->L alpha-->HII sequence in first heating, and reversible L beta<-->L alpha<-->HII phase sequences in subsequent heating and cooling scans. The peak areas and temperatures of the L beta-->L alpha transitions are practically identical, while the Lc-->L alpha and L alpha-->HII temperatures for 14-2,3-Gal are about 5 degrees C higher than the corresponding temperatures for 14-1,2-Glc. These data are interpreted in terms of a significantly greater stability and faster formation kinetics of the lamellar crystalline Lc phase of 14-1,2-Gal.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and phase behavior of a charged glycolipid (1,2-O-dialkyl-3-O-beta-D-glucuronosyl-sn-glycerol).

In order to investigate the effects of a net surface charge on the properties of glycolipid membranes, we have synthesized a glyceroglycolipid, 1,2-O-dialkyl-3-O-beta-D-glucuronosyl- sn-glycerol (GlcUA lipid), with saturated alkyl chains of varying length (14, 16, and 18 carbon atoms; 14-, 16-, and 18-GlcUA, respectively) and glucuronic acid with an ionizable 6-carboxyl group as polar residue. Aqueous dispersions of GlcUA lipids have been characterized by differential scanning calorimetry, densitometry, and X-ray diffraction methods as a function of pH. The carboxyl group deprotonation of apparent pK about 5.5 leads to a decrease of the melting temperatures by about 7 degrees C for all three compounds and to a chain-length-dependent reduction of the transition enthalpies by 0, 7, and 14% for 14-, 16-, and 18-GlcUA, respectively. The decrease of the transition temperature is consistent with current electrostatic concepts and models of charged membrane interfaces, but the chain-length-specific dependence of the enthalpy decrease with an increase of pH shows that the pH effects in GlcUA lipids are not of purely electrostatic origin. However, these effects appear to be simpler in some instances than corresponding effects in phospholipids with multiply ionizable head groups. For this reason, the lipids with the glucuronic acid head group appear to represent an appropriate model system for studies of net electric charge effects on the membrane properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereochemistry and size of sugar head groups determine structure and phase behavior of glycolipid membranes: densitometric, calorimetric, and X-ray studies.

The role carbohydrate moieties play in determining the structure and energetics of glycolipid model membranes has been investigated by small- and wide-angle X-ray scattering, differential scanning densitometry (DSD), and differential scanning microcalorimetry (DSC). The dependence of a variety of thermodynamic and structural parameters on the stereochemistry of the OH groups in the pyranose ring and on the size of the sugar head group has been studied by using an homologous series of synthetic stereochemically uniform glyceroglycolipids having glucose, galactose, mannose, maltose, or trimaltose head groups and saturated ether-linked alkyl chains with 10, 12, 14, 16, or 18 carbon atoms per chain. The combined structural and thermodynamic data indicate that stereochemical changes of a single OH group in the pyranose ring can cause dramatic alterations in the stability and in the nature of the phase transitions of the membranes. The second equally important determinant of lipid interactions in the membrane is the size of the head group. A comparison of lipids with glucose, maltose, or trimaltose head groups and identical hydrophobic moieties has shown that increasing the size of the neutral carbohydrate head group strongly favors the bilayer-forming tendency of the glycolipids. These experimental results provide a verification of the geometric model advanced by Israelachvili et al. (1980) [Israelachvili, J. N., Marcelja, S., & Horn, R. G. (1980) Q. Rev. Biophys. 13, 121-200] to explain the preferences lipids exhibit for certain structures. Generally galactose head groups confer highest stability on the multilamellar model membranes as judged on the basis of the chain-melting transition. This is an interesting aspect in view of the fact that galactose moieties are frequently observed in membranes of thermophilic organisms. Glucose head groups provide lower stability but increase the number of stable intermediate structures that the corresponding lipids can adopt. Galactolipids do not even assume a stable intermediate L alpha phase for lipids with short chain length but perform only Lc----HII transitions in the first heating. The C2 isomer, mannose, modifies the phase preference in such a manner that only L beta----HII changes can occur. Maltose and trimaltose head groups prevent the adoption of the HII phase and permit only L beta----L alpha phase changes. The DSD studies resulted in a quantitative estimate for the volume change associated with the L alpha----HII transition of 14-Glc. The value of delta v = 0.005 mL/g supports the view that the volume difference between L alpha and HII is minute.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers.

The structure and thermal behavior of hydrated and lyophilized dipalmitoylphosphatidylcholine (DPPC) multilayers in the presence of trehalose were investigated by differential scanning calorimetry and X-ray diffraction methods. Trehalose enters the aqueous space between hydrated bilayers and increases the interbilayer separation (from 0.36 to 1.37 nm in the different DPPC phases at 1 M trehalose). It does not affect the lipid chain packing and also the slow isothermal conversion at 4 degrees C of the metastable L beta' phase into the equilibrium crystalline Lc phase. Addition of trehalose leads to a slight upward shift (about 1 degrees C at 1 M trehalose) of the three phase transitions (sub-, pre-, and main transition) in fully hydrated DPPC while their other properties (enthalpy, excess specific heat, and transition width) remain unchanged. The effect of trehalose on the thermal behavior of DPPC multilayers freeze-dried from an initially completely hydrated state is qualitatively similar to that of water. These data support the "water replacement" hypothesis about trehalose action. It is suggested that trehalose prevents the formation of direct interbilayer hydrogen bonds in states of low hydration.

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases.

The phase transitions in fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases have been studied by lowangle time-resolved x-ray diffraction under conditions similar to those employed in calorimetry (scan rates 0.05-0.5 degrees C/min and uniform temperature throughout the samples). This approach provides more adequate characterization of the equilibrium transition pathways and allows for close correlations between structural and thermodynamic data. No coexistence of the rippled gel (P(beta')) and liquid-crystalline (L(alpha)) phases was found in the main transition of DPPC; rather, a loss of correlation in the lamellar structure, observed as broadening of the lamellar reflections, takes place in a narrow temperature range of approximately 100 mK at the transition midpoint. Formation of a long-living metastable phase, denoted by P(beta')(mst), differing from the initial P(beta') was observed in cooling direction by both x-ray diffraction and calorimetry. No direct conversion of P(beta')(mst) into P(beta') occurs for over 24 h but only by way of the phase sequence P(beta')(mst) --> L(beta') --> P(beta'). According to differential scanning calorimetry (DSC), the enthalpy of the P(beta')(mst)-L(alpha) transition is by approximately 5% lower than that of the P(beta')-L(alpha) transition. The effects of ethanol (Rowe, E. S. 1983. Biochemistry. 22:3299-3305; Simon, S. A., and T. J. McIntosh. 1984. Biochim. Biophys. Acta 773:169-172) on the mechanism and reversibility of the DPPC main transition were clearly visualized. At ethanol concentrations inducing formation of interdigitated gel phase, the main transition proceeds through a coexistence of the initial and final phases over a finite temperature range. During the subtransition in DPPC recorded at scan rate 0.3 degrees C/min, a smooth monotonic increase of the lamellar spacing from its subgel (L(c)) to its gel (L(beta')) phase value takes place. The width of the lamellar reflections remains unchanged during this transformation. This provides grounds to propose a "sequential" relaxation mechanism for the subgel-gel transition which is not accompanied by growth of domains of the final phase within the initial one.

Structure and phase behavior of hydrated mixtures of L-dipalmitoylphosphatidylcholine and palmitic acid. Correlations between structural rearrangements, specific volume changes and endothermic events.

Several new features of the phase diagram of L-dipalmitoylphosphatidylcholine (DPPC)/palmitic acid mixtures in excess water were established by means of static and time-resolved X-ray diffraction, densitometry and differential scanning calorimetry (DSC). At low temperatures, palmitic acid has a biphasic effect on the lamellar subgel phases: at concentrations below 5-6 mol%, it prevents formation of the DPPC subgel phase (Lc), while at higher contents (between about 40 and 90 mol%) another subgel phase (Lccom) is formed as a result of lipid co-crystallization at 1 DPPC: 2 palmitic acid stoichiometry. A crystalline palmitic acid phase separates from Lccom above 70-80 mol% of fatty acid. The Lccomphase transforms into a lamellar gel phase (L beta) in an endothermic transition centered at 38 degrees C. At high temperatures, the mixtures form hexagonal liquid-crystalline phase (HII) in the region of 60-70 mol% and an isotropic phase (I) at 90-100 mol% of palmitic acid. No coexistence of HII phase with the fluid lamellar phase of DPPC was observed at intermediate compositions (20 and 50 mol% of palmitic acid) but rather formation of a complex phase with non-periodic geometry characterized by molten chains and a broad, continuous small-angle scattering band. No evidence for fluid phase coexistence was found also at compositions between HII and I phases. The L beta--HII transition at 60-70 mol% of palmitic acids is readily reversible and two-state in both heating and cooling modes. It is characterized by the coexistence of initial and final phases with no detectable intermediates by time-resolved and static X-ray diffraction. The crystalline-isotropic transition in palmitic acid is two-state only in heating direction. On cooling, it is characterized by strong undercooling and gradually relaxing lamellar crystalline structures. The slowly reversible Lccom--L beta transition proceeds continuously through intermediate states. Although clearly discernible by both DSC and X-ray diffraction, it is not accompanied by specific volume changes.

Structural rearrangements during crystal-liquid-crystal and gel-liquid-crystal phase transitions in aqueous dispersions of dipalmitoylphosphatidylethanolamine. A time-resolved X-ray diffraction study.

The mechanism and kinetics of the crystal-liquid-crystal (Lc----L alpha) and gel-liquid-crystal (L beta----L alpha) transitions of the L-enantiomer and racemic dipalmitoylphosphatidylethanolamine have been examined in temperature scans and jumps using time-resolved X-ray diffraction methods. The Lc----L alpha transformations (at 66 degrees C for L-dipalmitoylphosphatidylethanolamine and 82 degrees C for DL-dipalmitoylphosphatidylethanolamine) were found to be two-state (first-order) processes characterised by co-existence of the initial Lc and final L alpha states during the transition with the absence of any detectable intermediates states. The transition mechanism involves firstly, disordering of the hydrocarbon chains which makes a major contribution to the transition enthalpy and secondly by a transition in the lamellar repeat spacing. The overall relaxation time of the Lc----L alpha transition of L-dipalmitoylphosphatidylethanolamine during temperature jumps of 4.5 degrees C/s was about 10 s. A gradual increase in the gel-state interchain spacing during the L beta----L alpha transitions of L- and DL-dipalmitoylphosphatidylethanolamine preceded a broadening of the wide-angle diffraction peak. There was a concomitant and continuous increase of the lamellar repeat spacing to values typical of the L alpha phase with increasing temperature. This sequence of events is completely reversible on cooling with a temperature hysteresis of 5-6 degrees C. The relaxation times of the L beta---L alpha transitions during jumps of 4.5 degrees C/s were about 2 s in both the heating and cooling directions.

Lamellar gel-lamellar liquid crystal phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from aqueous trehalose solutions. A real-time X-ray diffraction study.

The mechanism of the phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from fully hydrated gel phase (L beta') in the presence of trehalose has been investigated by real-time X-ray diffraction methods. Sequential diffraction patterns were recorded with an accumulation time of 3 s during heating and 1.2 s during cooling between about 20 and 80 degrees C. A transition is observed in the range 47-53 degrees C that involves structural events typical of a lamellar gel-lamellar liquid-crystal (L beta--L alpha) transformation. This transition is completely reversible with a temperature hysteresis of 2-3 degrees C and thereby resembles the main phase transition of fully hydrated dipalmitoylphosphatidylcholine multilayers. The mechanism of the transition from L beta to L alpha as seen in the wide-angle scattering profiles show that the sharp peak at about 0.41 nm, characteristic of the gel phase, broadens and shifts progressively to about 0.44 nm towards the end of the transition. A temperature jump of 6C degrees/s through the phase transition region of a freeze-dried dipalmitoylphosphatidylcholine: trehalose mixture (molar ratio 1:1) showed that the phase transition had a relaxation time of about 2 s which is similar to that of the main transition in the fully hydrated lipid. X-ray diffraction studies of the melting of dipalmitoylphosphatidylcholine freeze-dried from the lamellar-gel phase in the absence of trehalose showed a transition at above 70 degrees C. The low-angle diffraction data of phospholipid/trehalose mixtures are consistent with an arrangement of trehalose molecules in a loosely packed 'monolayer' separating bilayers of phospholipid. Trehalose appears to reduce the direct interbilayer hydrogen bond coupling thereby modifying the thermal stability and the phase transition mechanism of the bilayers.

Influence of head-group interactions on the miscibility of synthetic, stereochemically pure glycolipids and phospholipids.

Phase diagrams of binary mixtures of the glycoglycerolipids 1,2-di-O-tetradecyl-3-O-beta-D-galactosyl-sn-glycerol (14-Gal) and 1,2-di-O-tetradecyl-3-O-beta-D-glucosyl-sn-glycerol (14-Glc) with the phospholipids L-dimyristoylphosphatidylcholine (DMPC) and L-dimyristoylphosphatidylethanolamine (DMPE) were recorded by high-sensitivity differential scanning calorimetry and used for determination of the glycolipid-phospholipid miscibility in solid and liquid-crystalline states. As a consequence of a metastable behavior of both glycolipids and DMPE, the solid-state glycolipid/phospholipid miscibility was strongly dependent on the temperature prehistory of the samples. While DMPC and 14-Glc mix continuously, the other three binaries display extended regions of solid-solid-phase separation in the equilibrium low-temperature states. The DMPE/glycolipid phase diagrams were of clearly expressed eutectic type. Continuous solutions were formed in the liquid-crystalline and in the metastable solid phases of the mixtures. Simulations of the shape of the phase diagrams using the Bragg-Williams approximation showed certain deviations from ideal mixing in the liquid-crystalline continuous solutions. Since both glycolipids and phospholipids contain fully saturated fatty acids of equal chain length, their mixing properties were predominantly determined by the interactions between the lipid polar moieties, assuming the influence of ester or either linkages of the alkyl chains on the mixing parameters to be negligible. The clearly expressed differences in the mixing of 14-Glc and 14-Gal with phospholipids are most probably due to different hydrogen-bond networks formed by the glucosyl and galactosyl residues.

Mechanism and kinetics of the subtransition in hydrated L-dipalmitoylphosphatidylcholine.

The mechanism of the subtransitions (Lc to L beta') in L-dipalmitoylphosphatidylcholine bilayers in excess water has been investigated by time-resolved X-ray diffraction using synchrotron radiation. The temperature dependence of the diffraction patterns closely correlate with the asymmetric excess specific heat variation recorded by differential scanning calorimetry. During the subtransition two prominent wide-angle reflections, characteristic of the low-temperature crystalline phase, Lc, gradually change such that a sharp peak at a spacing of 0.430 nm decreases in intensity and ultimately disappears while a broader peak initially located at 0.375 nm progressively shifts to an eventual spacing of 0.410 nm. This behaviour is interpreted as a lateral deformation of the acyl chain packing subcell as the chains begin to rotate until a state is reached where the chains pack on a regular hexagonal array characteristic of the L beta phase. An increase in lamellar repeat distance from 6.0 to 6.4 nm takes place simultaneously with the acyl chain rearrangement at relatively low (5 K/min) as well as high (6 K/s) heating rates. As judged from the shape of the wide-angle peak, transformation to L beta' phase occurs some minutes after transition to the L beta phase. The X-ray data characterise the subtransition as a continuous (second order) phase transition in which a presumably orthorhombic subcell is transformed into a hexagonal subcell in a gradual process. In temperature jump experiments at 6 K/s between 0 degree C and 80 degrees C the relaxation time of the subtransition was found to be about 5 s while the relaxation time of the main gel to liquid-crystalline transition was about 2 s.

Effect of lipid admixtures on the L-dipalmitoylphosphatidylcholine subtransition.

The effect of lipid admixtures on the properties of the L-dipalmitoylphosphatidylcholine (L-DPPC) subtransition is investigated by using high-sensitivity differential scanning calorimetry. The four admixtures used are D-DPPC, L-dipalmitoylphosphatidylethanolamine (L-DPPE), cholesterol, and palmitic acid. In all cases the subtransition decreases in enthalpy until disappearance with increase of the admixture concentrations. About 5-7 mol% of D-DPPC or palmitic acid are sufficient for abolishment (without position shifts) of the subtransition, while, on addition of L-DPPE or cholesterol, it persists up to about 20 mol% of the admixture and its disappearance is accompanied by a slight shift to higher temperatures. These data are tentatively interpreted in terms of lateral mixing of L-DPPC and admixture as indicating compound formation with D-DPPC and palmitic acid, and clustering of L-DPPE and cholesterol.

A probability concept about size distributions of sonicated lipid vesicles.

The sonication procedure of preparation of small unilamellar vesicles is modelled as a process of uniform random fragmentation of the lipid aggregates. The vesicle size distribution evolving in this process is shown to be identical with the Weibull extremal probability distribution. Size histograms of sonicated small vesicles of various phospholipid composition were obtained by using electron microscopy (negative staining). Their successful simulation with Weibull curves shows that theory agrees with experiment. A similarly good agreement is found also with size histograms obtained by freeze-fracture of phosphatidylcholine-cholesterol vesicles (Van Venetië, R., Leunissen-Bijvelt, J., Verkleij, A.J. and Ververgaert, P.H.J.T. (1980) J. Microsc. 118, 401-408). This analysis allows a refinement of some earlier conclusions about the effect of cholesterol on the size of the sonicated vesicles. It follows from the theoretical model that the only intrinsic characteristic of the sonicated vesicles is the lower limit of their size. The other characteristics of the size distribution such as expectancy, dispersion, position and height of the maximum depend on the intensity of fragmentation. It is concluded that the size distribution of sonicated small vesicles is completely determined by the procedure of their preparation and, therefore, the condition of thermodynamic equilibrium between aggregated and monomeric lipid is irrelevant in this case.

The effect of nonideal lateral mixing on the transmembrane lipid asymmetry.

It is shown that the equilibrium transmembrane lipid asymmetry strongly depends on the degree of nonideality in the lateral mixing of the lipid components. In two-component bilayers the effect of nonideal lateral mixing is maximal for a given component at mole fractions of this component between 0.35 and 0.4. For asymmetry creating factors about 3 kT correcting for lateral nonidealities typical for lipids can increase as much as three times the transmembrane asymmetry. The relationship between lateral nonideality and transbilayer asymmetry is analysed in detail in the case of electrostatically induced asymmetry by using the Gouy-Chapman theory of electric double layers and the Bragg-Williams (regular solutions) approximation of nonideal lateral mixing. Two representative models are studied: (a) a single flat bilayer with a transmembrane electric potential difference applied on it; (b) two parallel membranes at short separation. In case (a), for transmembrane potentials of about 50-100 mV the introduction of nonideality corrections increases up to 40% the transmembrane asymmetry. In case (b), at physiological electrolyte concentrations the lipid asymmetry and, consequently, the effect of lateral nonideality become significant only at unrealistically small separations between the membranes. The surprisingly great influence of the lateral nonideality on the equilibrium transmembrane asymmetry suggests a significant role for this effect in determining the membrane molecular organization. A restricted lateral lipid miscibility might serve as a peculiar, but rather strong 'amplifier' of the transmembrane asymmetry. The qualitatively different asymmetries found in small unilamellar phosphatidylcholine-phosphatidylethanolamine vesicles of different fatty acid composition (Lentz, B.R. and Litman, B.J. (1978) Biochemistry 17, 5537-5543) can be reasonably well explained as an effect of the lateral nonideality. A hypothesis considering the transmembrane distributions of the major phospholipid species in erythrocytes as evolving from their lateral miscibilities is proposed.

Effect of ion concentration on phosphatidylethanolamine distribution in mixed vesicles.

The membrane of mixed phosphatidylcholine-phosphatidylethanolamine vesicles was found to be impermeable to the fluorescent label fluorescamine. Added to the vesicle solution, fluorescamine labels only the phosphatidylethanolamine molecules in the outer layer. The separation of labeled and free phospholipid by thin-layer chromatography permits the determination of the inner to outer phosphatidylethanolamine ratio. This ratio can be independently obtained by multiple sonication and the addition of fluorescamine, which results in a progressive increase of the labeled phosphatidylethanolamine. These two methods give identical results for the phospholipid distribution. The ratio of the outer to the total phosphatidylethanolamine decreases with the increase in the mole fraction of phosphatidylethanolamine, in agreement with the results published by Litman (Litman, B.J. (1973) Biochemistry 12, 2545-2554). It was also found that the lipid distribution is sensitive to changes of the electrolyte concentration in the aqueous phase. At high concentrations the distribution is close to the symmetrical one; a decrease in ionic strength results in preferential localisation of phosphatidylethanolamine molecules in the inner vesicle layer.

[Equilibrium surface charge distribution in phospholipid vesicles. III. Effect of nonelectrostatic factors]. / Ravnovesnoe raspredelenie poverkhnostnogo zariada v fosfolipidnykh vezikulakh. III. Vliianie neélektrostaticheskikh faktorov.

The different curvature of the two vesicle layers causes a difference in the standard free energies mu01 and mu02 of the lipid molecules in the inside and outside layers. In the present paper the influence of this difference on the equilibrium surface charge distribution is studied. The values of mu02--mu01 in the numerical calculations were in the range 0--3 kT. The results show the relative influence of electrostatic and non electrostatic forces on the equilibrium distribution of the charged lipid molecules. In the range of low surface charge density the charged molecules localize predominantly in the outside layer at low electrolyte concentrations, and in the inside layer at high electrolyte concentrations. A good agreement between the theoretical data and the experimental results of Berden et al. [3] about the phosphatidylserine distribution in lecithin-phosphatidylserine vesicles is demonstrated.

[Equilibrium surface charge distribution in phospholipid vesicles. II. Results of calculations]. / Ravnovesnoe raspredelenie poverkhnostnogo zariada v fosfolipidnykh vezikulakh. II. Rezul'taty raschetov.

The results of the calculations of the equilibrium potential and surface charge distribution in a vesicle with radii r1=55 A and r2=100 A are presented. The calculations were carried out for the electrolyte concentrations 100 mM, 10mM, 1mM and 0.1 mM. The digitally obtained solutions of the Poisson-Boltzman equation for the case of spherical symmetry were utilized in the exact solution of the problem. The predictions of the exact solution differ significantly from these of the linear approximation in the range of low electrolyte concentrations. The influence of the membrane dielectric permeability on the charge distribution is negligible.

[Equilibrium surface charge distribution in phospholipid vesicles. I. Method of calculation]. / Ravnovesnoe raspredelenie poverkhnostnogo zariada v fosfolipidnykh vezikulakh. I. Metod rascheta.

This paper presents a method of calculation of the surface charge equilibrium distribution between the two surfaces of a spherically closed phospholipid bilayer suspended in aqueous electrolyte solution. The net surface charge is supposed to be provided by the ionized polar groups of the phospholipid molecules. Its equilibrium distribution is found by minimization of the free electrostatic energy. The procedure of minimization utilizes the solution of the Poisson-Boltzmann equation which describes the double electric layers of the membrane and an expression for the membrane potential derived under the assumption of absence of charges in the membrane phase. An analytical solution of the problem in the range of validity of the linearized Poisson-Boltzman equation is obtained. It is shown that in this case an equilibrium transmembrane potential exists, and the surface charge density is greater at the outer surface of the vesicle.