T. E. Thompson

Sphingomyelins in bilayers and biological membranes

Abstract Sphingomyelin is one of the major lipids of the plasma membranes of mammalian cells. Together with phosphatidylcholine, the other choline-containing phospholipid, it makes up more than 50% of the total phospholipid in these membranes. In the plasma membranes of many cell types and over the course of diseases which affect cell membranes, although the total amount of those two lipids is constant, the membrane content of each of these phospholipids may vary greatly. Thus, it appears that these two choline-containing lipids are in certain measure interchangeable as membrane lipid components. This cannot, however, be the case because many of the physical characteristics of these molecules in bilayer systems are markedly different. Thus, variations in the relative amounts of sphingomyelin and phosphatidylcholine in bilayers and in biological membranes have profound effects on the system properties of the bilayer. Perhaps the most striking difference between phosphatidylcholines and sphingomyelins derived from biological membranes are the temperatures of the gel-liquid crystalline phase transition exhibited by both of these types of molecules in bilayers. Most sphingomyelins have their transition temperatures in the physiological temperature range, while almost all naturally occurring phosphatidylcholines are well above their transition temperature at 37°C. Thus, mixed phosphatidylcholine/sphingomyelin bilayers containing more than 50 mol% sphingomyelin exhibit a transition near 37°C, while those containing less than this amount show no transition in this temperature range. This characteristic is also reflected in the apparent microviscosity of the mixed bilayer at 37°C which increases with increasing content of sphingomyelin. The phase behavior of bilayers comprised of these two choline-containing lipids is strongly influenced by the addition of cholesterol. There is compelling evidence to suggest that the interaction between sphingomyelin and cholesterol is much stronger than it is between phosphatidylcholine and cholesterol. Thus, the microscopic phase configuration of simple bilayer systems is markedly affected by the relative concentration of sphingomyelin, phosphatidylcholine and cholesterol. By inference, the same situation exists in the bilayers of the plasma membranes of cells. The markedly different behavior of sphingomyelins and phosphatidylcholines in bilayer systems must reflect the differences in the molecular structures of these two classes of molecules. Although both molecular species have a polar region comprised of phosphorylcholine and a hydrophobic region comprised of two methylene chains, there are marked dissimilarities of structure elsewhere in the molecules. Phosphatidylcholines have two methylene chains of about equal length, while sphingomyelins have one methylene chain contributed by sphingosine which is of constant length. The other, contributed by the N- acyl group, is variable in length and can be up to 10 carbons longer than the sphingosine chain. This methylene chain length disparity in sphingomyelin is quite probably the basis, in part, for several interesting properties which are unique to bilayers composed of sphingomyelin. The generally lower degree of unsaturation of sphingomyelins relative to phosphatidylcholines also contributes to these differences. A third contributing factor is the difference in hydrogen bond-forming capability of the belt region which connects the polar and apolar regions of these molecules. The amide bond and hydroxyl group in this region of sphingomyelin can act as hydrogen bond donors while in phosphatidylcholine the carboxyl oxygens act as hydrogen bond acceptors. These differences in hydrogen bonding capabilities might be expected to be reflected in the interaction of these two lipids with other lipids in the bilayers and with membrane proteins. It is clear that the properties of bilayers comprised of these two superficially similar phospholipids reflect differences in molecular structure. Although the details of the relationships between molecular structure and properties and the system properties of bilayers comprised of these two phospholipid and cholesterols are not completely understood, much progress has been made. At the current level of this understanding, molecular explanations for certain of the physiologically important properties of biological membranes are beginning to emerge.

Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine

The non-ionic detergent octyl glucoside solubilizes a substantial amount of Streptococcus faecalis membrane protein without loss of the monitored enzyme activities. A secondary detergent, dioctanoyl phophatidycholine, appears to increase the yield of solubilized material. In addition, the effect of ionic strength indicates that it may be possible to selectively extract groups of membrane proteins by their characteristic solubility at different ionic strengths. The solubilized membrane-associated enzymes, ATPase and NADH dehydrogenase, enter polyacrylamide gels as distict species. Electrophoretic studies suggest that there are two membrane-associated ATPase in the Streptococcus faecalis, one which dissociates from the membrane in the absence of Mg-2+ ions and the other which remains particulate until solubilized by detergents. Octyl glucoside can be easily removed from a solution containing solubilized proteins and lipid by dialysis.

Capacitance, area, and thickness variations in thin lipid films

Abstract 1. 1. Thin lipid films are generally assumed to be homogeneous equilibrium structures with a definite chemical stoichiometry. However, occasional reports in the literature suggest that these assumptions may not be valid in all cases because of microlenses of solvent trapped in the films. We have studied in detail the specific capacitance of thin films formed from a chromatographically pure synthetic phospholipid in order to examine the validity of these assumption. 2. 2. The method of White ((1970) Biophys. J. 10, 1127–1148) for measuring the specific capacitance ( C m ) of planar lipid bilayers has been improved to allow C m to be determined with a precision of ±0.3% and accuracy of ±3.0%. Bilayer area ( A m ) is ascertained from photographs using a weight-area method. It is shown that calculations of A m based on measurements of film diameter using a microscope reticle are subject to a number of uncertainties which can greatly limit the precision and accuracy of area determinations. 3. 3. The total capacitance ( C T ), area, and specific capacitance of thin lipid films formed from 1,2-bisdihydrosterculoyl-3- sn -glycerophosphorylcholine in n -decane were measured as a function of time and applied voltage ( V A ). C T , A m , and C m generally varied with time and were non-reproducible. C m typically varied by 20% from film to film. A possible cause of these variations is microlenses of solvent trapped in the films and equations are derived which describe their effects on C m . It is concluded that the bilayer films studied must have a non-reproducible stoichiometry and a non-uniform thickness. The variations with time are probably a result of a disproportionation of n -decane (Andrews, D. M. and Haydon, D. A. (1968) J. Mol. Biol. 32, 149–150). 4. 4. C T of films in approximate equilibrium increases in the presence of an applied voltage ( V A ) due to an increase in both C m and A m . The dependence of C m on V A is accurately described by the equation C m = C 0 + sV A 2 . A similar, but approximate, equation is derived assuming the bilayer to be an elastic system of constant density which can be deformed by the force generated by the electric field.

PERMEABILITY OF DIMYRISTOYL PHOSPHATIDYLCHOLINE/DIPALMITOYL PHOSPHATIDYLCHOLINE BILAYER MEMBRANES WITH COEXISTING GEL AND LIQUID-CRYSTALLINE PHASES

The passive permeation of glucose and a small zwitterionic molecule, methyl-phosphoethanolamine, across two-component phospholipid bilayers (dimyristoyl phosphatidylcholine (DMPC)/dipalmitoyl phosphatidylcholine (DPPC) mixtures) exhibit a maximum when gel domains and fluid domains coexist. The permeability data of the two-phase bilayers cannot be fitted to single-rate kinetics, but are consistent with a Gaussian distribution of rate constants. In pure DMPC and DPPC as well as in their mixtures, at the temperature of the maximum excess heat capacity, the logarithm of the average permeability rate constants are linearly correlated with the mole fraction of DPPC in the total system. In addition, in the 50:50 mixture, the excess heat capacity values as well as the apparent fractions of interfacial lipid correlate with the logarithm of the excess permeabilities in the two-phase region. These results suggest that small polar molecules can cross the membrane at the interface between gel and fluid domains at a much faster rate than through the homogeneous phases; the acyl chains located at the domain interface experience lateral density fluctuations that are inversely proportional to their average length, and large enough to allow rapid transmembrane diffusion of the solute molecules. The distribution of the permeability rate constants may reflect temporal and spatial fluctuations of the lipid composition at the phase boundaries.

Percolation and diffusion in three-component lipid bilayers: effect of cholesterol on an equimolar mixture of two phosphatidylcholines

The lateral diffusion of a phospholipid probe is studied in bilayers of binary mixtures of dimyristoylphosphatidylcholine (DMPC)/cholesterol and distearoylphosphatidylcholine (DSPC)/cholesterol and in the ternary system DMPC/DSPC/cholesterol using fluorescence recovery after photobleaching. An approximate phase diagram for the ternary system, as a function of temperature and cholesterol concentration, was obtained using differential scanning calorimetry and the phase diagrams of the binary systems. This phase diagram is similar to those of the phospholipid/cholesterol binary mixtures. In bilayers where solid and liquid phases coexist, the diffusion results are interpreted in terms of phase percolation. The size of the liquid-phase domains is estimated using percolation theory. In the ternary system, addition of cholesterol up to approximately 20 mol% shifts the percolation threshold to lower area fractions of liquid, but the size of the liquid-phase domains does not change. Above approximately 20 mol% cholesterol, the liquid phase is always connected. The size of solid-phase domains clusters is estimated using a model recently developed (Almeida, P.F.F., W.L.C. Vaz, and T.E. Thompson. 1992. Biochemistry. 31:7198-7210). For cholesterol concentrations up to 20 mol%, the size of solid-phase domain units does not change. Beyond 20 mol%, cholesterol causes the size of the solid units to decrease.

Monte Carlo Simulation of Two-Component Bilayers: DMPC/DSPC Mixtures*

In this paper, we describe a relatively simple lattice model of a two-component, two-state phospholipid bilayer. Application of Monte Carlo methods to this model permits simulation of the observed excess heat capacity versus temperature curves of dimyristoylphosphatidylcholine (DMPC)/distearoylphosphatidylcholine (DSPC) mixtures as well as the lateral distributions of the components and properties related to these distributions. The analysis of the bilayer energy distribution functions reveals that the gel-fluid transition is a continuous transition for DMPC, DSPC, and all DMPC/DSPC mixtures. A comparison of the thermodynamic properties of DMPC/DSPC mixtures with the configurational properties shows that the temperatures characteristics of the configurational properties correlate well with the maxima in the excess heat capacity curves rather than with the onset and completion temperatures of the gel-fluid transition. In the gel-fluid coexistence region, we also found excellent agreement between the threshold temperatures at different system compositions detected in fluorescence recovery after photobleaching experiments and the temperatures at which the percolation probability of the gel clusters is 0.36. At every composition, the calculated mole fraction of gel state molecules at the fluorescence recovery after photobleaching threshold is 0.34 and, at the percolation threshold of gel clusters, it is 0.24. The percolation threshold mole fraction of gel or fluid lipid depends on the packing geometry of the molecules and the interchain interactions. However, it is independent of temperature, system composition, and state of the percolating cluster.

Organization of ganglioside GM1 in phosphatidylcholine bilayers

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.

Organization of the glycosphingolipid asialo-GM1 in phosphatidylcholine bilayers.

An affinity purified monovalent ferritin conjugate of Ricinus communis agglutinin (RCA 60) is used with freeze-etch electron microscopy to study the ultrastructural localization of the glycosphingolipid asialo-GM1 in multilamellar phosphatidylcholine liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes containing up to 20 mol% asialo-GM1 and quenched below the main transition temperature show a striking linear localization of ferritin-RCA 60 between phospholipid ridges. The glycosphingolipid localization is similar to that postulated for up to 20 mol% cholesterol in pure phosphatidylcholine bilayers by Copeland, B.R. and McConnell, H.M. (Biochim. Biophys. Acta, 599, 95-109 (1980)). Above the main phase transition temperature, asialo-GM1 appears to be organized into clusters, especially in palmitoyloleoylphosphatidylcholine (POPC) liposomes. This clustered distribution of glycosphingolipids seen above the phase transition temperature suggests that this type of lipid may exhibit compositional domain structure in biological membranes.

Physical properties of bilayer membranes formed from a synthetic saturated phospholipid in n-decane

A pure phosphatidyl choline containing branched-chain, saturated acyl substituents has been prepared by a novel synthesis. Bilayer membranes formed from this non-autoxidizable, pure phospholipid are very stable and have an interfacial tension of 1.5 ± 0.1 dyne/cm at 24°. Both the electrical resistance and capacitance of the bilayer are pH dependent. The resistance has a maximum value of about 109 Ω/cm2 in the pH range 4–6. The specific capacitance has a minimum value of 0.38 μF/cm2 at pH 4 with a limiting value of 0.43 μF/cm2 at high pH. The basis for the pH dependence of these electrical parameters is unknown.

Properties of a specific glycolipid transfer protein from bovine brain.

A transfer protein specific for glycolipids has been isolated from bovine brain. As judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, the protein is 68% pure and has a molecular weight of 20 000. Three different assays were employed to study the proteins specificity and glycolipid binding properties. The protein transferred several different neutral glycosphingolipids and ganglioside GM1 equally well, but failed to accelerate phosphatidylcholine or sphingomyelin intervesicular movement. The proteins ability to interact with glycolipids was strongly influenced by the physical properties of the matrix phospholipid in which the glycolipids reside. Both the phase state of the phospholipid matrix and bilayer curvature affected glycolipid intervesicular transfer rates. Protein binding to phospholipid vesicles containing either tritium-labeled or pyrene-labeled glucosylceramide could not be demonstrated by density gradient centrifugation or fluorescence energy transfer measurements, respectively. A specific association of the transfer protein for pyrene-labeled glucosylceramide was found when the fluorescence emission of the pyrene excimer-to-monomer ratio was measured suggesting that a portion of the fluorescent glycolipid was being sequestered from the phospholipid vesicles and was binding to the freely soluble protein.