Y. Barenholz

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.

[91] Fluorescent probe: Diphenylhexatriene

Publisher Summary This chapter discusses the fluorescent probe—diphenylhexatriene. Depolarization of fluorescence has proved to be a very reliable technique with which to characterize the thermotropic and dynamic aspects of the hydrophobic regions of lipid bilayers and lipoproteins. Fluorescence analysis of dynamic systems can be performed either with time-dependent or steady-state type anisotropy measurements. The former approach can yield information concerning the heterogeneity of the lifetime of the fluorophore in the system, allowing in some cases a resolution of fluorophore subpopulations. This technique also allows an evaluation of the hindrance of the probe motion because of the degree of order of the phospholipid acyl side chains. Fluorescence polarization is measured by exciting the fluorophore with monochromatic light through a polarizer whose polarization axis is oriented vertically to the light path and the emission intensity is detected through an analyzer, whose polarization axis is oriented sequentially parallel to and perpendicular to the polarization axis of the exciting light.

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.

Fluorescence properties of cholestatrienol in phosphatidylcholine bilayer vesicles

The fluorescent sterol delta 5,7,9,(11)-cholestatrien-3 beta-ol (cholestatrienol) was incoporated into 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. Previously another fluorescent sterol, dehydroergosterol (F. Schroeder, Y. Barenholz, E. Gratton and T.E. Thompson. Biochemistry 26 (1987) 2441), was used for this purpose. However, there is some concern that dehydroergosterol may not be the best analogue for cholesterol. Fluorescence properties of cholestatrienol in POPC SUV were highly sensitive to cholestatrienol purity. The fluorescence decay of cholestatrienol in the POPC SUV was analyzed by assuming either that the decay is comprised of a discrete sum of exponential components or that the decay is made up of one or more components distribution of lifetimes. The decay for cholestatrienol in POPC SUV analyzed using distributions had a lower chi 2 value and was described by a two-component Lorentzian function with centers near 0.86 and 3.24 ns, and fractional intensities of 0.96 and 0.04, respectively. Both distributions were quite narrow, i.e., 0.05 ns full-width at half-maximum peak height. It is proposed that the two lifetime distributions are generated by separate continua of environments for the cholestatrienol molecule described by different dielectric constants. In the range 0-6 mol% cholestatrienol, the cholestatrienol underwent a concentration-dependent relaxation. This process was characterized by red-shifted absorption and maxima and altered ratios of absorption and fluorescence excitation maxima. Fluorescence quantum yield, lifetime, steady-state anisotropy, limiting anisotropy and rotational rate remained constant. In contrast, in POPC vesicles containing between 6 and 33 mol% cholestatrienol, the fluorescent cholestatrienol partially segregated, resulting in quenching. Thus, below 6 mol% cholestatrienol, the cholestatrienol appeared to behave in part as monomers exposed to some degree to the aqueous solvent in a sterol-poor domain within POPC bilayers. Since the lifetime did not decrease above 6 mol% cholestatrienol, the fluorescence at high mol% values of cholestatrienol was due to cholestatrienol in the sterol-poor domain. The fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy of cholestatrienol in the sterol-poor domain decreased to limiting, nonzero values while the rotational rate increased to a limiting value. Thus, the sterol-poor domain became more disordered when it coexisted with the sterol-rich domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental changes in plasma membrane fluidity in chick embryo heart

1. Decreases in the rate of transport of sugars (facilitated transport), amino acids (active transport), and urea (simple diffusion) occur in chick embryo heart during development. This work considers the possibility that changes in the plasma membrane fluidity during development contribute to the observed changes in transport activities. 2. Technics were developed for subcellar fractionation of chick embryos and adult chickens. 3. The depolarization of the fluorescence of 1,6-diphenylhexatriene was used to estimate the fluidity of the lipid portion of plasma membrane enriched fractions of hearts from chick embryos at various stages of development and from adult hearts. 4. There is a pattern of decreasing membrane viscosity as development proceeds. Between 5-6 days and 10 days of embryonic life a 20% decrease in viscosity of the plasma membrane-enriched fraction occurs. Between 10 and 20 days of embryonic life there is no significant change in viscosity. Between 20 days of development (1 day before hatching) and adulthood there is a further 55% decrease in plasma membrane viscosity. 5. It is proposed that the changes in membrane fluidity observed may contribute to developmental changes in membrane transport activities, but other factors must also be involved.

Permeability and integrity properties of lecithin-sphingomyelin liposomes

The properties of multibilayered liposomes formed from mixtures of sphingomyelin and phosphatidylcholine in varying mole ratio (all containing one mole dicetylphosphate per 10 moles of phospholipids) have been studied. The principal findings are: (1) Over the range 0 to 1 mole fraction sphingomyelin the liposomes exhibit multibilayer structure as visualized by electron microscopy using negative staining. (2) The two phospholipids differ in their interaction with dicetylphosphate in a bilayer structure. In mixtures of the two the effect of sphingomyelin is dominant. (3) The ability of sphingomyelin to form osmotically active liposomes depends on its fatty acids composition. (4) Liposomes of all mole fractions of sphingomyelin are osmotically active if the C24: 1 fatty acid content of sphingomyelin exceeds 10% of the total acyl residues. The degree of osmotic activity, however, depends upon the molar ratio between the two phospholipids. The highest initial rate of water permeability was found for lecithin liposomes. The maximal change of volume by osmotic gradients was obtained for liposomes composed of 1:1 lecithin to sphingomyelin (mole ratio). (5) Permeability to glucose increased with increasing lecithin mole fraction. (6) Liposomes composed of 1:1 lecithin to sphingomyelin have the largest aqueous volume per mole of phospholipid as measured by glucose trapping. (7) The osmotic fragility of liposomes made of sphingomyelin is higher than for those made of lecithin but the highest osmotic fragility was obtained for liposomes containing lecithin and sphingomyelin in 1:1 molar ratio. (8) When the temperature is abruptly lowered to about 2 degrees C, lipsomes formed from phosphatidylcholine release about 20% of trapped glucose during a transient increase in permeability. Liposomes containing 0.5 mole fraction sphingomyelin release about 30% of the trapped glucose under these conditions. Liposomes composed of sphingomyelin alone do not exhibit this phenomenon.

The relations between the composition of liposomes and their interaction with triton X-100

Abstract The interaction of Triton X-100 with phospholipid bilayers of multilamellar liposomes formed from mixtures of spinal cord sphingomyelin and egg yolk lecithin in various mole ratios (all containing 1 mole of dicetylphosphate per 10 moles of phospholipid) was studied. The results indicate that the process is time dependent and is much slower than the formation of simple micelles. The time to reach the final equilibrium state is dependent on the SPM to PC mole ratio, on the Triton to phospholipid mole ratio, and on the Tirton concentration. Titration with increasing Triton concentration shows that the behavior of Triton is biphasic for all the various lipid compositions tested. For low Triton to phospholipid mole ratio there is no mass formation of mixed micelles; in addition, the Triton seems to radically increase the leakage of glucose without reducing the turbidity. This range is limited by a turning point where most of the phospholipids and about half of the Triton coprecipitate. Above this Triton to phospholipid mole ratio formation of mixed Triton-phospholipid micelles occurred followed by a drastic decline in turbidity. This turning point as well as the exact profile of the Triton effect are strongly related to the SPM:PC mole ratio. The higher the mole fraction of SPM in the membrane, the less Triton is required to reach the turning point and to cause a complete solubilization. These effects can be explained by tighter packing and stronger phospholipid-phospholipid interactions imposed by SPM and expressed as apparent microviscosity which increases upon increasing the mole fraction of SPM in the bilayer.

Spontaneous transfer between phospholipid bilayers of dehydroergosterol, a fluorescent cholesterol analog

The spontaneous interbilayer transfer of dehydroergosterol, a fluorescent cholesterol analog, was examined using small unilamellar phospholipid vesicles. The kinetic data were best fit by an equation of the form Aexp (-kt) + B. Qualitatively, the general trend of the half-time for transfer and the base values (B) obtained for dehydroergosterol resemble the corresponding values obtained in the earlier studies of cholesterol transfer. However, quantitative differences, which reflect the molecular structure of the sterol, were observed. Acrylamide quenching performed on the donor vesicles at different stages of the transfer indicated that a time-dependent organization of DHE within the vesicles occurs.

Lateral Organization of Pyrene-labeled Lipids in Bilayers as Determined from the Deviation from Equilibrium between Pyrene Monomers and Excimers

In lipid bilayers, pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner. The aromatic amine N,N-diethylaniline (DEA), which has a high membrane-to-medium partition coefficient, quenches the monomers only, and therefore it is expected that under conditions in which the monomers are in equilibrium with the excimers due to the mass law, the Stern-Volmer coefficient (K) for monomers (M), defined as K, should be identical to that of the excimer (E), defined as K, and K/K = 1.0. This is indeed the case for pyrene and pyrene valerate in egg phosphatidylcholine small unilamellar vesicles. However, for pyrene decanoate and pyrene dodecanoate in these vesicles, and for N-[12-(1-pyrenyl)dodecanoyl]sphingosylphosphocholine in a matrix of either N-stearoyl sphingosylphosphocholine or 1-palmitoyl-2-oleoyl phosphatidylcholine, K < K. This can be explained either by the existence of (a) two subpopulations of excimers, one in fast equilibrium with the monomers and the other, related to ground-state protoaggregates of pyrene lipids; (b) two monomer subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both quenched by DEA, but only one of which produces excimers. The good agreement between the photophysical processes determined by steady state and time-resolved measurements supports the third explanation for the bilayers containing pyrene phospholipids. It also suggests that the main factors determining the immiscibility of pyrene lipids in phospholipid bilayers are the temperature, the difference in the gel-to-liquid-crystalline phase transition temperature (ΔT) between the matrix and the pyrene lipid, and the structural differences between the matrix lipid and the pyrene-labeled lipid. These results indicate that the K/K ratio can serve as a very sensitive tool to quantify isothermal microscopic immiscibility in membranes. This novel approach has the following advantages: applicability to fluid phase immiscibility, requirement of a relatively low mol fraction of pyrene lipids, and conceivably, applicability to biological membranes.

A simple method for the synthesis of cholesterol esters in high yield

A simple and convenient synthesis of cholesterol esters from cholesterol and fatty acid anhydrides is described. The high, reproducible yield of cholesterol ester and the small excess of fatty acid used makes this method attractive for the preparation of cholesterol esters using difficultly obtained fatty acids.