Showing papers on "Membrane lipids published in 1981"

Glycosphingolipids in Cellular Interaction, Differentiation, and Oncogenesis

Abstract: The idea that glycosphingolipids (or, briefly, glycolipids) are ubiquitous components of plasma membrane and display cell type-specific patterns perhaps stemmed from the classical studies on glycolipids of erythrocyte membranes.(1,2) Subsequently, plasma membranes of various animal cells were successfully isolated and analyzed; all were characterized by their much higher content of glycolipid than was found in intracellular membranes.(3–8) It is generally assumed that glycolipids are present at the outer leaflet of the plasma membrane bilayer, although this assumption is based only on experiments with surface-labeling by galactose oxidase-NaB[3H]4 of intact and lysed erythrocyte membranes and inside-out vesicles.(9,10) Obviously, further extensive studies of the organization of glycolipid in other eukaryotic cell membranes are necessary. Interestingly, the majority of neutral glycolipids present in plasma membranes are cryptic (see Section 4.2.1).

The stimulation of inositol lipid metabolism that accompanies calcium mobilization in stimulated cells: defined characteristics and unanswered questions.

Abstract: It now appears to be generally agreed that the 'phosphatidylinositol response', discovered in 1953 by Hokin & Hokin, occurs universally when cells are stimulated by ligands that cause an elevation of the ionized calcium concentration of the cytosol. The initiating reaction is almost certainly hydrolysis of an inositol lipid by a phosphodiesterase. Phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate all break down rapidly under such circumstances. However, we do not yet know which of these individual reactions is most closely coupled to receptor stimulation, nor do we know where in the cell it occurs. With many stimuli, inositol phospholipid breakdown is closely coupled to occupation of receptors and appears not to be a response to changes in cytosol [Ca2+]: this provoked the suggestion that it may be a reaction essential to the coupling between activation of receptors and the mobilization of Ca2+ within the cell. In a few situations, however, it appears probable that inositol lipid breakdown can occur as a result of the rise in cytosol [Ca2+] that follows receptor activation: such observations gave rise to the alternative opinion that inositol lipid breakdown cannot be related to stimulus-response coupling at calcium-mobilizing receptors. It now seems likely that these two views are too rigidly polarized and that some cells probably display both receptor-linked and Ca2+-controlled breakdown of inositol lipids. Both may sometimes occur simultaneously or sequentially in the same cell.

Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval.

Abstract: A survey of species differences in the duration of capacitation, T, has revealed that they closely correlate with sperm cholesterol/phospholipid mole ratios, R : T = 8R - 1 (r2 = 0.97, in which r is Pearson's correlation coefficient). Because uterine cells displayed low relative cholesterol concentrations, spermatozoa evidently experience a negative external cholesterol gradient (positive phospholipid gradient) during capacitation. A decrease in sperm R-value is suggested, therefore, to accompany capacitation. The idea received strong support from a kinetic analysis of capacitation intervals, based on the rate of cholesterol efflux from sperm cells in utero. Lipid-binding serum proteins in uterine fluid are attributed with removing a sterol barrier to the Ca2+-facilitated membrane fusion that initiates the acrosome reaction. Tight cell junctions prevent permeation of the male generative tract by these proteins (capacitation factors). Furthermore, seminal plasma contains a decapacitation factor, identified as a membrane vesicle (cholesterol donor) component of this fluid, that reverses capacitation. Initiation of the sperm acrosome reaction among mammals could be the first fusion process found to be physiologically modulated through the membrane bilayer cholesterol level.

Specific gangliosides function as host cell receptors for Sendai virus.

Abstract: The ability of specific gangliosides to function as host cell receptors for Sendai virus was investigated by using Madin-Darby bovine kidney cells which become resistant to infection upon treatment with Vibrio cholerae sialidase. Sialidase-treated cells were incubated for 20 min at 37 degrees C with individual, highly purified gangliosides containing homogeneous carbohydrate moieties and then inoculated with virus for 10 min. Susceptibility of the cells to infection was monitored by hemagglutination titer of the virus produced 48 hr after inoculation. Incubation of the cells with gangliosides containing the sequence NeuAc alpha 2,3Gal beta 1,3GalNAc (i.e., GD1a, GT1b, and GQ1b) fully restored susceptibility to infection to the cells. However, the ganglioside GQ1b in which the sequence ends with two sialic acids in a NeuAc alpha 2,8NeuAc linkage instead of a single sialic acid as in GD1a and GT1b, was effective as a receptor at a concentration 1/100th that of any of the other gangliosides tested. Incubation with gangliosides similar in structure to GD1a, GT1b, and GQ1b but lacking the sialic acid attached to the terminal galactose (i.e., GM1 and GD1b) had no effect. The results from control experiments in which gangliosides were incubated at 0 degrees C with cells or in which trypsin was used to remove gangliosides adsorbed to cells were consistent with the premise that the gangliosides must actually insert into the cellular membrane to function as Sendai virus receptors. Addition of 4 X 10(6) molecules of 14C-labeled GD1a per cell made the cells fully susceptible to infection. Analysis of the ganglioside content of cell membranes showed that gangliosides GD1a, GT1b, and GQ1b are natural components of these cells and are present in quantities sufficient to act as receptors. These results demonstrate that gangliosides with the proper carbohydrate sequence, such as GD1a, GT1b, and GQ1b, function as natural receptors for Sendai virus in host cells.

Abnormalities in Membrane Phospholipid Organization in Sickled Erythrocytes

Abstract: In contrast to the wealth of information concerning membrane phospholipid asymmetry in normal human erythrocytes, very little is known about membrane phospholipid organization in pathologic erythrocytes. Since the spectrin-actin lattice, which has been suggested to play an important role in stabilizing membrane phospholipid asymmetry, is abnormal in sickled erythrocytes, we determined the effects of sickling on membrane phospholipid organization. We used two enzymatic probes: been venom phospholipase A2 and Staphylococcus aureus sphingomyelinase C, which do not penetrate the membrane and react only with phospholipids located in the outer leaflet of the bilayer. Our results suggest that the distribution of glycerophospholipids within the membrane of sickled cells is different from that in nonsickled cells. Compared with the normal erythrocyte, the outer membrane leaflet of the deoxygenated, reversibly sickled cells (RSC) and irreversibly sickled cells (ISC) was enriched in phosphatidyl ethanolamine in addition to containing phosphatidyl serine. These changes were compensated for by a decrease in phosphatidyl choline in that layer. The distribution of sphingomyelin over the two halves of the bilayer was unaffected by sickling. In contrast to ICS, where the organization of phospholipids was abnormal under both oxy and deoxy conditions, reoxygenation of RSC almost completely restored the organization of membrane phospholipids to normal. These results indicate that the process of sickling induces an abnormality in the organization of membrane phospholipids to normal. These results indicate that the process of sickling induces an abnormality in the organization of membrane lipids in RSC which become permanent in ISC.

Ethanol and the Physical Properties of Brain Membranes: Fluorescence Studies

Abstract: Synaptic plasma membranes and myelin were prepared from mouse brain and their physical properties were evaluated by using the fluorescent probe molecules 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-aminopyrene. The absorption-corrected fluorescence output and fluorescence polarization of DPH (a probe of the membrane core) were decreased when synaptic plasma membranes were exposed in vitro to low concentrations (10-20 mM) of ethanol. Fluorescence of 1-aminopyrene (a probe of the membrane surface) was affected only by high ethanol concentrations. Ethanol did not alter the excitation or emission maxima of DPH in synaptic plasma membranes, which indicates that it did not alter polarity in the vicinity of the probe. Compared with the intact synaptic plasma membranes, DPH fluorescence in myelin membranes and in lipids extracted from myelin or synaptic plasma membranes was less sensitive to the effects of ethanol. Analysis of fluorescence polarization at various temperatures indicated that ethanol altered the phase behavior of membrane lipids. These results indicate that low, physiologically relevant concentrations of ethanol selectively fluidize the hydrophobic core of synaptic membranes. The sensitivity of intact synaptic membranes is discussed in terms of lipid-protein interactions disrupted by ethanol.

Matrix control of protein diffusion in biological membranes

Abstract: Lateral diffusion coefficients of fluorescently labeled lipids and integral membrane proteins were determined in the membranes of normal and spectrin-deficient spherocytic mouse erythrocytes by the technique of fluorescence redistribution after photobleaching The results were used to generate a mathematical description of a matrix-control model of membrane protein diffusion In the spherocytic cells, which lack the principal components of the cytoskeletal matrix of normal cells, the diffusion coefficients of lipid (15 +/- 05 X 10(-8) cm2/s) and protein (25 +/- 06 X 10(-9) cm2/s) differ only by a factor of 6, close to the difference predicted on the basis of size by the two-dimensional bilayer continuum model of Saffman and Delbruck [Saffman, P G l& Delbruck, M (1975) Proc Natl Acad Sci USA 72, 3111-3113] In contrast, the membranes of normal cells show a lipid diffusion coefficient (14 +/- 05 X 10(-8) cm2/s) that is some 300-fold greater than that of the membrane proteins (45 +/- 08 X 10(-11) cm2/s) Analysis of these results, based on the hypothesis that protein diffusion in normal membranes is sterically hindered by a labile matrix, yields an effective matrix surface viscosity consistent with the viscoelastic mechanical properties of the membranes Thus, a relationship is established between the deformation characteristics of the membrane and the lateral mobility of proteins suspended in the membrane

Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles.

Abstract: Native staphylococcus aureus alpha-toxin is secreted as a hydrophilic polypeptide chain of Mr 34,000. The presence of deoxycholate above the critical micellar concentration induced the toxin monomers to self-associate, forming ring or cylindrical oligomers. The oligomers were amphiphilic and bound detergent. In deoxycholate solution, the protein-detergent complexes exhibited a sedimentation coefficient of 10.4 S. A Mr of 238,700 was determined by ultracentrifugation analyses at sedimentation equilibrium. Because quantitative detergent-binding studies indicated a protein/detergent ratio of approximately 5:1 (wt/wt), the protein moiety in each protein-detergent complex was determined to be approximately Mr 200000, corresponding to a hexamer of the native molecule. The amphiphilic toxin hexamers were ultrastructurally indistinguishable from the cytolytic, annular toxin complexes that form on and in biological target membranes. They bound lipid and could be incorporated into artificial lecithin lipid vesicles. The transition of toxin protein molecules from a hydrophilic monomer to an amphiphilic oligomer through self-association has thus been shown to be inducible solely through contact of the native protein molecules with an appropriate amphiphilic substrate.

Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at pH 5.2

Abstract: The molecular mechanism of hemolysis and fusion by influenza virus in acidic media was studied. First, the effect of trypsin treatment on the activity of fibroblast-grown influenza virus was studied. The results showed that the split form of viral hemagglutinin, HA1 and HA2, but not the precursor, is responsible for the activity. Second, the interaction of egg-grown influenza virus, which contains the split hemagglutinin, with lipid liposomes was studied by spin labeling and electron microscopy. Phospholipid transfer from the viral envelope to the lipid bilayer membrane occurred within 30 s at pH 4.5-5.4. The transfer is largely independent of the lipid composition and the crystalline vs. liquid/crystalline state of the membrane. Virus-induced lysis of liposomes also took place rapidly in the same pH range. Envelope fusion with liposomes occurred at pH 5.2 but not at pH 7.0. These characteristic interactions were similar to those between influenza virus and erythrocytes reported previously. On the other hand, hemagglutinating virus of Japan did not interact with liposomes at neutral pH. These results suggest that protonation of the NH2-terminal segment of the HA2 form causes interaction of the segment with the lipid core of the target cell membrane, leading to hemolysis and fusion.

Membranes and phospholipids of liver mitochondria from chronic alcoholic rats are resistant to membrane disordering by alcohol

Abstract: Using the spin probe 5-doxylstearic acid, we studied the structural perturbations of rat liver mitochondrial membranes produced by exposure to ethanol in vitro and by chronic ethanol feeding. The addition of ethanol in vitro to mitochondria from control animals appears to "fluidize" the membranes, as evidenced by a pronounced decrease in the order parameter. By contrast, in membranes from rats fed ethanol chronically, there was no effect on the order parameter. This resistance of the mitochondrial membranes from chronically intoxicated animals to the fluidizing effect of ethanol probably results from a change in the composition of the phospholipids, because the same differential response to ethanol was observed upon using vesicles of mitochondrial phospholipids extracted from control and chronically treated rats. In the presence of 0.025--0.1 M ethanol, a range that prevails in the blood of chronic alcoholics, the order parameter of mitochondrial membranes from rats fed ethanol was comparable to that of control membranes without ethanol in vitro. Analysis of extracted mitochondrial phospholipids showed that the cardiolipin from ethanol-fed animals had fatty acyl residues that are more saturated than those of controls. These findings point to the underlying molecular mechanism of our previous observation that mitochondria from chronic alcoholic rats are more resistant to uncoupling by ethanol at physiological temperature [Rottenberg, H., Robertson, D. E. & Rubin, E. (1980) Lab. Invest. 42, 318--326]. We suggest that an adaptive change in the phospholipid composition leads to structural alterations, which result in increased resistance to disruption of mitochondrial membranes by ethanol. These changes in lipid composition and structure may explain many, if not all, of the mitochondrial abnormalities that have been previously reported to result from chronic ethanol intoxication.

Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging.

Abstract: The addition of malondialdehyde to erythrocytes in vitro causes a decrease in bands 1 and 2 of spectrin and an increase in high molecular weight protein polymers. Additionally, this agent causes the formation of fluorscent chromolipids characteristic of those produced during the peroxidation of endogenous membrane phospholipids. These same alterations in proteins and lipids are observed in the membranes of older cells fractionated from freshly drawn blood and in the membranes of reticulocytes induced by treatment of animals with phenylhydrazine, but not in reticulocytes induced by bleeding. The former reticulocytes have a much shorter half-life in the circulation than do either normal erythrocytes or reticulocytes produced consequent to bleeding. These experiments and the apparent paradox of "young" reticulocytes with short half-lives suggest that the in vivo polymerization of membrane proteins consequent to radical-induced peroxidation of membrane lipids may contribute to the altered rheological behavior and hence to the splenic sequestration of cells. They also suggest that increases in intrinsic membrane rigidity due to lipid peroxidation, malondialdehyde, and protein polymerization may be a common feature of both aging in normal erythrocytes and in the accelerated aging that accompanies the administration of radical-generating, hemolytic agents. However, it is cautioned that other polymerization reactions involving disulfides, calcium, or direct radical attack on protein monomers may also be important determinants of the visco-elastic properties of erythrocyte membranes.

Lipid--protein multiple binding equilibria in membranes.

Abstract: Phospholipids at the lipid--protein interface of membrane proteins are in dynamic equilibrium with fluid bilayer. In order to express the number of binding sites (N) and the relative binding constants (K) in terms of measurable quantities, the equilibrium is formulated as an exchange reaction between lipid molecules competing for hydrophobic sites on the protein surface. Experimental data are reported on two integral membrane proteins, cytochrome oxidase and (Na,-K)-ATPase, reconstituted into defined phospholipids. Electron spin resonance measurements on reconstituted preparations of beef heart cytochrome oxidase in 1,2-dioleoyl-sn-3-phosphatidylcholine containing small quantities of the spin-labeled phospholipid 1-palmitoyl-2-(14-proxylstearoyl)-sn-3-phosphatidylcholine (PC*) gave a linear plot of bilayer/bound PC* vs. the lipid/protein ratio as predicted by the theory, with K congruent to 1 and N = 40 (normalized to heme aa3). This demonstrates that the spin-label moiety attached to the hydrocarbon chain does not significantly perturb the binding equilibria. In the second experimental system, (Na,K)-ATPase purified from rectal glands of Squalus acanthias was reconstituted with defined phosphatidylcholines as the lipid solvent and spin-labeled phospholipids with choline or serine head groups (PC*, PS*) as the solute. The (Na,K)-ATPase has a larger number of lipid binding or contact sites (N = 60-65 per alpha 2 beta 2 dimer) and exhibits a detectably larger average binding constant for the negatively charged phosphatidylserine than for the corresponding phosphatidylcholine. These results show that a multiple equilibria, noninteracting site binding treatment can account for the behavior of lipids exchanging between the protein surface and the lipid bilayer. Selective sites among a background of nonselective sites are experimentally detectable as a change in the measured relative binding constant.

Insertion of influenza M protein into the viral lipid bilayer and localization of site of insertion.

Abstract: Recent studies with isolated M protein from influenza virus have shown that the protein has a high affinity for lipid The ability of M to partition into lipid vesicles merely by shaking vesicles and M together is suggestive evidence that the protein could be interacting with the lipid in the virus particle A more direct analysis was carried our here to determine whether M is in contact with the viral lipid in situ, by using the photoactivatable hydrophobic probe, pyrenesulfonyl azide Covalent linkage of this probe to M indicated that a segment of M residues with in the virus membrane in contact with the lipid bilayer M inserted into lipid vesicles at two locations on the molecule A major insertion into lipid occurred in the middle of the molecule where a large cluster of 20 hydrophobic and neutral amino acids occurs A second insertion occurred approximately one fourth in from the amino terminus, where a smaller segment of 13 uncharged amino acids is found Confirmation that M inserted into lipid at these locations came also from results with cyanogen bromide fragments of M Of the 12 to 13 fragments produced, 3 specifically bound to lipid vesicles These were the first, second, and third contiguous segments beginnings at the amino terminus and containing the two hydrophobic areas noted above

Phosphatidate as a molecular link between depolarization and neurotransmitter release in the brain

Abstract: Phosphatidate, a neuronal phospholipid, stimulated the uptake of calcium by nerve terminals isolated from the striatum of rat brain. This effect was not produced by other phospholipids or glycolipids. Phosphatidate, but not other phospholipids, evoked the release of [3H] dopamine from striatal synaptosomes. The magnitude of both effects was similar to that observed after chemical depolarization of the nerve terminals. These results show that phosphatidate is the only membrane lipid component that acts as a functionally competent ionophore and support the suggestion that phosphatidate may serve as a link between depolarization and neurotransmitter release in the brain.

The asymmetric transmembrane distribution of phosphatidylethanolamine, phosphatidylserine, and fatty acids of the bovine retinal rod outer segment disk membrane

Abstract: The transmembrane distribution of the major aminophospholipids in the bovine retinal rod outer segment disk membrane, phosphatidylethanolamine and phosphatidylserine, was determined using a novel pair of permeable and impermeable covalent modification reagents. The values for the percentages of phosphatidylethanolamine and phosphatidylserine in the outer monolayer were calculated from a simple expression which takes into account the leakage of impermeable reagent into the disk lumen as monitored by the extent of labeling of lysine entrapped in the lumen. We infer from our results that at least 73 to 87% of the disk phosphatidylethanolamine and 77 to 88% of the disk phosphatidylserine are in the outer disk membrane monolayer. The fatty acid composition of the inner aminophospholipids is slightly more saturated than the outer aminophospholipids. Calculations using the lateral surface areas occupied by the disk membrane lipids suggest that 65 to 100% of the disk phosphatidylcholine is on the inner membrane surface. Since the disk phosphatidylcholine is also somewhat more saturated than the phosphatidylethanolamine and phosphatidylserine of the outer monolayer, the total inner membrane monolayer fatty acid composition is more saturated than that of the outer monolayer fatty acid composition.

The headgroup conformation of phospholipids in membranes.

Abstract: An important feature which determines the physical state of biological membranes is the great variety of individual lipids occurring in different amounts in the composition of the lipid matrices. One may assume that by this means nature has created conditions which optimize the function of the individual highly specialized protein systems in the membranes of different cells and organelles. Present membrane research is far away from presenting an unambiguous proof for this suggestion. Nevertheless for several reconstituted membrane systems, where one integral membrane protein was embedded in one or more kinds of lipids, it was demonstrated that changes in the spectrum of the hydrocarbon chain conformations and their rates of segmental motions (which determine the fluidity of the bilayer) induces variations in the distribution of the proteins in the plane of the membrane and in their function. Large changes in these properties occur for most phospholipids in bilayers during a first-order phase transition between a so-called gel state and a liquid crystalline state. One clear example for the influence of the lipid state on a protein distribution in the bilayer was given by Kleemann and McConnell (1976) in the reconstitution of Ca-ATPase in bilayers of dimyristoyllecithin. Electron micrographs showed the proteins aggregated in the gel state and more randomly distributed in the liquid crystalline state. Considering lipid influence on membrane protein function, the work of Overath, Schairer and Stoffel (1970) gives an example of the way in which the lipid state influences sugar transport through membranes of Escherichia coli which were enriched in some lipid species. These examples illustrate the influence of the chain conformation on membrane proteins.

I-Kk and H-2Kk antigens are shed as supramolecular particles in association with membrane lipids.

Abstract: The form of 125I-labeled membrane alloantigens (I-Ak and H-2Kk) and immunoglobulin M shed by murine splenic lymphocytes labeled by lactoperoxidase-catalyzed radioiodination was determined by gel chromatography and ultracentrifugation. I-Ak and H-2Kk antigens are shed as particles ranging in size from 3 X 10(5) to 2 X 10(6), whereas membrane IgM is shed as an individual unit consonant with its native size of 180,000. The effects of detergents on the size of shed I-Ak and H-2Kk and the coprecipitation of (3H) lipids with these proteins suggest that I-Ak and H-2Kk are shed in association with local membrane lipids as small membrane-derived vesicles, whereas membrane IgM is shed without detectable associated lipid.