Phospholipid exchange between bilayer membranes

The mode of interaction of aqueous dispersions of phospholipid vesicles is investigated. The vesicles (average diameter 950 Å) are prepared from total lipid extracts of Escherichia coli composed of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. One type of vesicle contains $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$, the other type $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$ as predominant acyl chain component. The vesicles show order?disorder transitions at transition temperatures, $\text{T}{}_{\mathrm{<mtext>t</mtext>}}\text{= 42°}\text{C}$ and $\text{T}{}_{\mathrm{<mtext>t</mtext>}}\text{= 29°}\text{C}$, respectively. A mixture of these vesicles is incubated at 45° C and lipid transfer is studied as a function of time using the phase transition as an indicator. The system reveals the following properties: Lipids are transferred between the two vesicle types giving rise to a vesicle population where both lipid components are homogeneously mixed. Lipid transfer is asymmetric, i.e. $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$-containing lipid molecules appear more rapidly in the $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$-containing vesicles than vice versa. At a given molar ratio of the two types of vesicles the rate of lipid transfer is independent of the total vesicle concentration. It is concluded that lipid exchange through the water phase by way of single molecules or micelles is the mode of communication of these negatively charged lipid vesicles.     

Phospholipid exchange between bilayer membrane vesicles.

The turbidity of lipid vesicles, freshly prepared by sonicating purified dimyristoyllecithin (DML) in dilute KCl solutions, was measured as a function of time at various temperatures. A sharp maximum in the rate of increase of turbidity is found just above the crystal:liquid-crystal phase transition temperature (Tm). The initial rate of turbidity increase is first order with respect to DML concentration. Electron and light microscopy reveal large vesicles which are not present before incubation or after incubation at temperatures far from the Tm. When temperature, rather than time, is the independent variable, a sharp drop in turbidity is seen at the Tm. The magnitude of this drop and the temperature at which it occurs were used to measure the rate of lipid transfer between vesicles composed of different lipids. A mixture of DML vesicles and dipalmitoyllecithin (DPL) vesicles exhibits sharp drops in turbidity at 24 and 41 degrees, the corresponding Tm's. With time, the magnitude of the transition at 24 degrees decreases while that which was originally at 41 degrees moves to lower temperatures and increases in magnitude. At equilibrium there is a single transition at 32.5 degrees characteristic of vesicles composed of equimolar DPL and DML. The rate at which equilibrium is approached increases at around 24 degrees and again around 41 degrees. These observations indicate that vesicles are in equilibrium with monomolecular lipid, the concentration of the latter being higher the shorter the lipid acyl group or the smaller the vesicle. DML molecules are therefore lost from small vesicles to large vesicles (DML system) or lost from DML vesicles to DML-DPL vesicles (mixed system). When DML vesicles containing a few percent brain gangliosides were studied, different behavior was observed; the initial rate of increase of turbidity becomes second order in lipid concentration, and the rate constant increases with increasing concentrations of KCl. The kinetic order, coupled with the fact that electrolyte reduces intervesicle electrostatic repulsion, argues that in this situation the mechanism of vesicle growth requires vesicle collision.     

Kinetics of phospholipid exchange between bilayer membranes.

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469,311--325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, k--, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are kP- = (0.86 +/- 0.05) - 10(-5) S-1 and ke- = (1.09 +/- 0.13) - 10(-6) s-1 for phospholipid molecules with trans-delta 9-hexadecenoate and trans-delta 9-octadecenoate, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

Kinetics of phospholipid exchange between bilayer membranes

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469, 311–325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, $\text{k}{}_{\mathrm{?}}$, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>P</mtext>}}\text{= (0.86 ± 0.05) · 10}{}^{\mathrm{?5}}\text{s}{}^{\mathrm{?1}}$ and $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>E</mtext>}}\text{= (1.09 ± 0.13) · 10}{}^{\mathrm{?6}}\text{s}{}^{\mathrm{?1}}$ for phospholipid molecules with $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$ and $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

Phospholipid surface bilayers at the air-water interface. III. Relation between surface bilayer formation and lipid bilayer assembly in cell membranes.

Lipid bilayer assembly in cell membranes has been simulated with total lipid extracts from human red blood cells and from mesophilic and thermophilic bacteria grown at several temperatures. Aqueous dispersions of these natural lipid mixtures form surface bilayers, a single bimolecular lipid state, but only at the growth temperature of the source organism. Thus, a single isolated bilayer state forms spontaneously in vitro from lipids that are available in vivo at the growth temperature of the cell. Surface bilayers form at a specific temperature that is a function of hydrocarbon chain length and degree of fatty acid unsaturation of the phospholipids; this property is proposed as an essential element in the control of membrane lipid composition.     

Phosphatidylcholine exchange between the boundary lipid and bilayer domains in cytochrome oxidase containing membranes.

A phospholipid spin label, 16-doxylphosphatidylcholine, is employed in a study of lipid--protein interactions in cytochrome oxidase containing membranes. Two methods are used to label the membranous cytochrome oxidase: dispersion in cholate with subsequent detergent removal, and fusion with vesicles of the pure phospholipid label in the absence of detergent. A fraction of the label is immobilized, which is calculated to fall in the range of 0.17--0.21 mg of phospholipid/mg of protein (0.15--0.19 after correction for lipids not extracted by chloroform--methanol). This narrow range of values is independent of methods of labeling, protein isolation, and lipid depletion within experimental error. When labeling by fusion is utilized, the patches of pure phosphatidylcholine spin label diffuse in the plane of the bilayer, become diluted, and demonstrate exchange with bound phospholipid. These observations are evidence that boundary lipid, as reflected by the partitioning of the phosphatidylcholine label, is in equilibrium with adjacent bilayer regions and that it consists of a relatively constant amount of phospholipid associated with the hydrophobic portion of the protein.     

Lipid exchange between membranes.

The exchange of lipid molecules between vesicle bilayers in water and a monolayer forming at the water surface was investigated theoretically within the framework of thermodynamics. The total number of exchanged molecules was found to depend on the bilayer curvature as expressed by the vesicle radius and on the boundary condition for exchange, i.e., whether during exchange the radius or the packing density of the vesicles remains constant. The boundary condition is determined by the rate of flip-flop within the bilayer relative to the rate of exchange between bi- and monolayer. If flip-flop is fast, exchange is independent of the vesicle radius; if flip-flop is slow, exchange increases with the vesicle radius. Available experimental results agree with the detailed form of this dependence. When the theory was extended to exchange between two bilayers of different curvature, the direction of exchange was also determined by the curvatures and the boundary conditions for exchange. Due to the dependence of the boundary conditions on flip-flop and, consequently, on membrane fluidity, exchange between membranes may partially be regulated by membrane fluidity.     

Interactions between digitonin and bilayer membranes

The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form in flat lamellar sheets. In contrast, when the complex is formed in a bilayer membrane, the membrane is deformed into corrugations of hemitubules. The polarity of the deformations formed in bilayer membranes is highly correlated with the direction of entry of digitonin into the membrane. We suggest that the morphology of digitonin/cholesterol hemitubules is dependent upon the complex being formed within a bilayer and, in addition, is not correlated with asymmetry of cholesterol concentration across the membrane.     

Interaction between bending and tension forces in bilayer membranes.

A theoretical analysis is presented of the bending mechanics of a membrane consisting of two tightly-coupled leaflets, each of which shears and bends readily but strongly resists area changes. Structures of this type have been proposed to model biological membranes such as red blood cell membrane. It is shown that when such a membrane is bent, anisotropic components of resultant membrane tension (shear stresses) are induced, even when the tension in each leaflet is isotropic. The induced shear stresses increase as the square of the membrane curvature, and become significant for moderate curvatures (when the radius of curvature is much larger than the distance between the leaflets). This effect has implications for the analysis of shape and deformation of freely suspended and flowing red blood cells.     

Membrane fusion. Transfer of phospholipid molecules between phospholipid bilayer membranes

Fusion of phosphatidylcholine (PC) vesicles and of PC-phosphatidylserine (PS) vesicles has been studied using spin-labeled PC and PS. Analysis of ESR spectra indicated transfer of phospholipid molecules between phospholipid vesicles at the instant of membrane contact by vesicular collision. The transfer rate of PC was not greatly affected by the presence of the anionic lipid in the membranes. The rate of PC transfer between PS-PC vesicles was nearly the same as that of PS transfer. Calcium ion greatly enhanced the transfer of phospholipid molecules between PS-PC vesicles. The enhancement of PS transfer occurred instantaneously. The phospholipid transfer is related to the fusion of vesicles.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

Retinol transfer across and between phospholipid bilayer membranes

The transfer of retinol across and between bilayer membranes was studied in vitro using unilamellar liposomes and erythrocytes. Transmembrane movement of retinol in phospholipid bilayer membranes was a spontaneous and rapid process with a halflife of less than 30 s. Retinol transfer between liposomes and between liposomes and erythrocytes was also a spontaneous and rapid process with a halflife of less than 10 min. The results suggest that retinol transport in the cell might not need the participation of specific transfer proteins.     

Interactions between tricyclic antidepressants and phospholipid bilayer membranes

Participation of electrostatic and other noncovalent interactions in the binding of tricyclic antidepressants (TCAs) to the lipid bilayers was estimated from pH-dependencies of imipramine, desipramine, amitriptyline and nortriptyline binding to the lipid bilayers prepared from different phospholipids, both electroneutral and acidic. The binding was studied using a radioligand binding assay. It was found that the membrane phospholipid composition and methylation of the acyl side chain of TCA has a decisive effect on participation of particular noncovalent interactions in the binding. Apparent high-affinity binding of TCAs to the phosphatidylcholine or phosphatidylethanolamine membranes are achieved mainly by incorporation of uncharged drug molecules into the hydrophobic core of the bilayers. Van der Waals forces and hydrophobic effect are responsible for this binding. Both charged and uncharged drug molecules bind to phosphatidylserine membranes, therefore coulomb- or ion-induced dipole interactions play a role in these binding. Different spatial distribution of charged residues within the interface causes different electrostatic interactions between charged TCAs and vesicles formed from phosphatidylserine and phosphatidylinositol. The data supports the hypothesis under which TCAs could have effect on affective disorders partially via binding to the lipid part of the membrane and following changes of lipid-protein interactions.     

Transfer of cholestane spin label between lipid bilayer membranes and its molecular motion in membranes.

The relation between the molecular motion of a steroid in lipid membranes and the transfer rate between membranes was examined using radioactive cholestane spin label. Order parameters of the molecule were determined in bilayers composedof dipalmitoylglycerophosphocholine or egg yolk phosphatidylcholine at various temperatures. The line widths of the ESR signal of the cholestane spin label in membranes, which depend upon the rate of molecular axial rotation in the membranes, were also measured. The temperature dependences of these two parameters and of the transfer rate suggest a close correlation between the rate of molecular axial rotation and the transfer rate.     

Fusion of synaptic vesicle membranes with planar bilayer membranes.

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.     

Planar bilayer membranes from pure lipids.

Planar lipid bilayer membranes are formed from mixtures of pure lipids in the absence of non-biological solvents. The solventless bilayers are characterized by a large specific capacitance (586-957 nF/cm2) comparable to that of cell membranes but considerably greater than that of conventional lipid/decane bilayers. Hydrocarbon solvents, such as n-alkanes or squalene, thicken the bilayer. Membrane dielectric thickness is used as an indicator of bilayer lipid composition. For membranes made from pure monoglyceride/triglyceride mixtures the thickness of the solventless lipid bilayer is independent of both the chain length (11-22 carbons) and mol fraction (0.1-0.9) of triglyceride in the bulk mixture. In contrast, the thickness of the bilayer (2.0-3.3 nm) depends strongly upon the length (16-24 carbons) of the monoglyceride component. Molecular volume considerations lead to the conclusion that the bulk lipid mixture disproportionates to yield bilayer membranes composed of nearly pure monoglyceride. The dielectric thickness of the monoglyceride bilayer is consistent with the notion that the lipid fatty acyl chains are fluid.