Incorporation of exogenous glycosphingolipids in plasma membranes of cultured hamster cells and concurrent change of growth behavior

Globoside added to culture medium was taken up by NIL cells and accumulated as a component of plasma membrane. This was evidenced by the recovery of ³H-labelled globoside from plasma membrane fractions and by the higher chemical quantity of globoside found in NIL cells cultured in medium containing globoside. Concomitantly the following changes in growth behavior were manifested: a reduction in growth rate due to an extended prereplicative phase and a reduced saturation density which may result from changed adhesive properties of cells.     

Effects of cations on dipalmitoyl phosphatidylcholine/cholesterol/water systems.

X-ray diffraction studies have been made on the effects of cations upon the dipalmitoyl phosphatidylcholine/water system, which originally consists of a lamellar phase with period of 64.5 A and of excess water. Addition of 1 mM CaCl2 destroys the lamellar structure and makes it swell into the excess water. The lamellar phase, however, reappears when the concentration of CaCl2 increases: a partially disordered lamellar phase with the repeat distance of 150-200 A comes out at the concentration of about 10 mM, the lamellar diffraction lines become sharp and the repeat distance decreases with increasing CaCl2 concentration. A small amount of uranyl acetate destroys the lameellar phase in pure water. MgCl2 induces the lamellar phase of large repeat distance, whereas LiCl, NaCl, KCl, SrCl2 and BaCl2 exhibit practically no effect by themselves. Addition of cholesterol to the phosphatidylcholine bilayers tends to stabilize the lamellar phase. The high-angle reflections indicate that molecular arrangements in phosphatidylcholine bilayers change at CaCl2 concentrations around 0.5 M. The bilayers at high CaCl2 concentration seem to consist of two phases of pure phosphatidylcholine and of equimolar mixture of phosphatidylcholine and cholesterol.     

Interactions of cholesterol esters with phospholipids: cholesteryl myristate and dimyristoyl lecithin.

The ternary phase diagram of cholesteryl myristate--dimyristoyl lecithin--water has been determined by polarizing light microscopy, scanning calorimetry, and x-ray diffraction. Hydrated dimyristoyl lecithin forms a lamellar liquid--crystalline phase (L alpha) at temperatures greater than 23 degrees C into which limited amounts of cholesteryl myristate (less than 5 wt. %) can be incorporated. The amount of cholesterol ester incorporated is dependent upon the degree of hydration of the L alpha phase. Below 23 degrees C dimyristoyl lecithin forms ordered hydrocarbon chain structures (L beta' and P beta') which do not incorporate cholesterol ester. Comparison with other phospholipid--cholesterol ester--water phase diagrams suggests the following general principles: i) the incorporation of cholesterol ester occurs only into liquid crystalling phospholipid bilayers, ii) the extent of incorporation is temperature-dependent, with increasing amounts of cholesterol ester being incorporated at higher temperatures, and iii) unsaturated cholesterol esters induce increased disordering of the phospholipid bilayers.     

The ternary phase diagram of lecithin, cholesteryl linolenate and water: phase behavior and structure

The ternary phase diagram of cholesteryl linolenate-egg lecithin-water has been determined by polarizing light microscopy, calorimetry and X-ray diffraction at 23 °C. Hydrated lecithin forms a lamellar liquid-crystalline structure into which small amounts of cholesteryl linolenate are incorporated. The maximum incorporation of cholesterol ester into this lamellar structure varies with the degree of hydration. Increasing the water concentration from 10 to 15% (w/w) increased the limiting molar ratio of cholesteryl linolenate to lecithin in the lamellar phase from 1:50 to 1:22. At intermediate concentrations (15 to 30% water) the cholesteryl linolenate:lecithin ratio remains constant at 1:22. When water is increased to 42.5%, the maximum water content in the lamellar phase, the molar ratio decreased to 1:32. At low water concentrations the cholesterol ester appears to be entirely in the apolar region of the lecithin bilayer, while at higher water concentrations the ester groups of cholesteryl linolenate may be located at the lipid-water interface. At high water concentrations the ester appears to disorder the alkyl chains of the lecithin, giving rise to a thinner lipid layer and an increased surface area per lipid molecule when compared to the lecithin-water system in the absence of cholesteryl linolenate.The lamellar phase is the only phase (except at water concentrations less than 5%) in which all three components mutually interact. All mixtures of the three components having compositions outside the one-phase (lamellar) zone produce additional phases of cholesteryl linolenate or water, or both. Between 23 °C and 60 °C only minor changes in the phase diagram are observed.     

Deuterium magnetic resonance studies of phospholipid bilayers

L-α-dipalmitoyl lecithin is selectively deuterated at two different chain positions. The residual quadrupole splittings of the corresponding phospholipid bilayers are measured by means of deuterium magnetic resonance and evaluated in terms of the segmental order parameters. The results are briefly compared with other esr and nmr investigations of lipid bilayers.     

The interaction of alpha-tocopherol with bilayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine

The effect of alpha-tocopherol on the structure and phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylcholine was examined by real-time synchrotron X-ray diffraction and freeze-fracture electron microscopic methods. X-ray scattering intensity was recorded from mixed aqueous dispersions of phospholipid with 2.5, 5, 10 and 20 mol% alpha-tocopherol during temperature scans at 3 degrees /min between -25 and 10 degrees C. A ripple structure is induced by the presence of alpha-tocopherol that coexists with the ripple phase characteristic of the pure phospholipid in mixtures containing 2.5 mol% alpha-tocopherol but completely replaces it in mixtures containing greater proportions of alpha-tocopherol. Freeze-fracture replicas of dispersions containing 5 mol% alpha-tocopherol indicate a ripple phase with a periodicity of about 9 nm. Increasing amounts of alpha-tocopherol result in a progressive reduction in temperature of the gel to liquid-crystal phase transition and broadening of the transition. Two lamellar phases coexist in the liquid-crystal state, one with a spacing of 6.4 nm assigned to an alpha-tocopherol-enriched lamellar structure and the other with a lamellar repeat of 6.1 nm corresponding to bilayers of pure phospholipid.     

Interactions of helical polypepetide segments which span the hydrocarbon region of lipid bilayers. Studies of the gramicidin A lipid-water system.

The polypeptide gramicidin A in a dimeric form is considered to form a helical structure which spans the hydrocarbon region of lipid bilayers. In the present investigation it is used as a model for the interactions of the polypeptide segments of transmembrane proteins within the hydrocarbon region of the lipid bilayers of biomembrane structures. A variety of physical techniques (X-ray diffraction, differential scanning calorimetry, optical and electron microscopy, Raman and electron spin resonance spectroscopy) are applied to a study of the interactions of this polypeptide within the phospholipid bilayers of dimyristoyl and dipalmitoyl lecithins in water, at temperatures both above and below the main endothermic phase transition of the pure lipids.Above the transition temperature of the lipid, the Raman studies show that the polypeptide perturbs the fluid lipid environment and causes a marked decrease in the number of gauche isomers of the lipid hydrocarbon chains, even at quite low relative molar concentrations of the polypeptide to lipid (1:150). At concentrations of phospholipid to polypeptide of less than 5:1, the electron spin resonance studies show the existence of two lipid regions within the bilayer. One region corresponds to the relatively fluid lipid region normally observed at these temperatures and the other to a relatively rigid lipid region. The latter is considered to arise from clusters of the polypeptide in which some of the lipid is entrapped.Below the lipid phase transition temperature, the pretransition endotherm observed with pure lipid-water systems is removed by small molar concentrations of the polypeptide (1:50) and the rippled appearance observed in freeze-fracture electron micrographs with pure dimyristoyl lecithin-water dispersions is replaced by a smooth appearance.The main lipid phase transition becomes broadened by the presence of increasing amounts of the polypeptide within the lipid bilayer as indicated by calorimetry, and electron spin resonance spectroscopy. The enthalpy of the lipid transition decreases linearly with increasing amounts of the polypeptide until, with dipalmitoyl lecithin, a concentration of approximately 20 lipids per polypeptide is reached. This is considered to correspond to the onset of an aggregation process which produces localised polypeptide-lipid clusters within the plane of the membrane.At concentrations of polypeptide less than five lipids per polypeptide, freezefracture electron microscopy shows the presence of liposomes with smooth fracture faces. At higher polypeptide concentrations, sheet-like structures are observed with smooth fracture faces.When a mixed lipid-water system (dilauroyl and dipalmitoyl lecithin) containing low concentrations of the polypeptide is slowly cooled, the calorimetric evidence shows that the polypeptide moves preferentially into the lower melting region of the bilayer, whereas at higher polypcptide eoncentrations a mixing of the two lipids takes place.The various results are discussed to provide insight pertinent to the organisation, interactions, aggregation properties, boundary layer and packing arrangements of helical polypeptides and proteins in reconstituted systems and natural biomembranes.     

Structural requirements for the formation of ordered lipid multibilayers--a spin probe study

Spin probes were intercalated in oriented films formed from a series of lipids to study their ability to form ordered bilayers. Factors found to be important are the size and charge of the headgroup, the number, length, and degree of unsaturation of the acyl chains, the presence of cholesterol, and the type and concentration of ions in the aqueous phase. Unsaturated diglycerides and monoglycerides did not form bilayers; saturated monoglycerides formed well-ordered bilayers above their transition temperatures and cholesterol allowed the formation of ordered bilayers at lower temperatures. Saturated diglycerides did not form bilayers at temperatures at which the probe was dissolved in the system. The addition of calcium ions increased the anisotropy of films formed from a mixture of lecithin, cholesterol, and phosphatidic acid, but disrupted films formed from phosphatidylserine. Caution must be exercised in interpreting electron spin resonance spectra of films since films of tightly packed lipids tend to exclude spin probes and preparations which are clearly lamellar manifest a high degree of disorder within the bilayers.     

Effects of cations on dipalmitoyl phosphatidylcholine/cholesterol/water systems

X-ray diffraction studies have been made on the effects of cations upon the dipamitoyl phosphatidylcholine/water system, which originally consists of a lamellar phase with period of 64.5 Å and of excess water. Addition of 1 mM CaCl₂ destroys the lamellar structure and makes it swell into the excess water. the lamellar phase, however, reappears when the concentration of CaCl₂ increases: a partially disordered lamellar phase with the repeat distance of 150–200 Å comes out at the concentration of about 10 mM, the lamellar diffraction lines become sharp and the repeat distance decreases with increasing CaCl₂ concentration. A small amount of uranyl acetate destroys lamellar phase in pure water. MgCl₂ induces the lamellar phase of large repeat distance, whereas LiCl, NaCl, KCl, SrCl₂ and BaCl₂ exhibit practically no effect by themselves. Addition of cholesterol to the phosphatidylcholine bilayers tends to stabilize the lamellar phaseThe high-angle reflections indicate that molecular arrangements on phosphatidylcholine bilayers change at CaCl₂ concentrations around 0.5 M. The bilayers at high CaCl₂ concentration seem to consist of two phases of pure phosphatidylcholine and of equimolar mixture of phosphatidylcholine and cholesterol.     

Phase behavior and structure of aqueous dispersions of sphingomyelin

The phase behavior of bovine brain sphingomyelin in water has been determined by polarizing light microscopy, differential scanning calorimetry, and X-ray diffraction. Lamellar phases, in which water is intercalated between sheets of lipid molecules arranged in the classical bilayer fashion, are present over much of the phase diagram. An order-disorder transition separates the high temperature, liquid crystalline, lamellar phase from a more ordered lamellar phase at low temperatures. The hydration characteristics of sphingomyelin are similar to the structurally related lecithin in that only limited amounts of water are incorporated above and below the transition. Above the transition at 47 degrees C, a maximum of 35% by weight of water can be incorporated between the lipid bilayers, the total thickness at maximum hydration being 60.2 A, the lipid thickness 38 A, and the surface area per lipid molecule at the interface 60 A(2). Water in excess of 35% by weight is present as a separate phase. Below the phase transition, at 25 degrees C a maximum of 42% by weight of water may be incorporated between the lipid bilayers. On increasing the hydration, the lamellar repeat distance increases from 63.5 A to a limiting value of 76 A. Within this hydration range the calculated lipid thickness decreases from 63.5 to 42.5 A, and the surface area per lipid molecule increases from 36.1 to 53.6 A(2). Although these changes may be accounted for by a structure in which the hexagonally packed ordered hydrocarbon chains tilt progressively with respect to the normal to the bilayer plane on increasing hydration, it is possible that changes in other more complex lamellar structures may be responsible for these variations in lipid thickness and surface area.     

In-plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids

SAXS/WAXS studies were performed in combination with freeze fracture electron microscopy using mixtures of a new Gemini catanionic surfactant (Gem16-12, formed by two sugar groups bound by a hydrocarbon spacer with 12 carbons and two 16-carbon chains) and the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) to establish the phase diagram. Gem16-12 in water forms bilayers with the same amount of hydration water as DPPC. A frozen interdigitated phase with a low hydration number is observed below room temperature. The kinetics of the formation of this crystalline phase is very slow. Above the chain melting temperature, multilayered vesicles are formed. Mixing with DPPC produces mixed bilayers above the corresponding chain melting temperature. At room temperature, partially lamellar aggregates with local nematic order are observed. Splitting of infinite lamellae into discs is linked to immiscibility in frozen state. The ordering process is always accompanied by dehydration of the system. As a consequence, an unusual order-disorder phase transition upon cooling is observed.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.     

On the localization of ubiquinone in phosphatidylcholine bilayers

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05-0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [13C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the 13C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (P beta') phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.     

Globoseries glycosphingolipids of human meconium

Globoside and an extended globoseries glycosphingolipid with a blood group H determinant were isolated from pooled human meconia and structurally characterized by mass spectrometry, proton NMR spectroscopy, and degradational techniques using GC and GC-MS analyses. Both species contained mainly phytosphingosine and hydroxy fatty acids characteristic for human intestinal epithelial cells. With the same techniques also minor amounts of globoside with sphingosine and nonhydroxy fatty acids and a novel globoseries tetraglycosyl ceramide with a terminal N-acetylglucosamine were isolated and structurally characterized.     

Measurement and modification of forces between lecithin bilayers.

We probe in two different ways the competing attractive and repulsive forces that create lamellar arrays of the phospholipid lecithin when in equilibrium with pure water. The first probe involves the addition of low molecular weight solutes, glucose and sucrose, to a system where the phospholipid is immersed in a large excess of water. Small solutes can enter the aqueous region between bilayers. Their effect is first to increase and then to decrease the separation between bilayers as sugar concentration increases. We interpret this waxing and waning of the lattice spacing in terms of the successive weakening and strengthening of the attractive van der Waals forces originally responsible for creation of a stable lattice. The second probe is an "osmotic stress method," in which very high molecular weight neutral polymer is added to the pure water phase but is unable to enter the multilayers. The polymer competes for water with the lamellar lattice, and thereby compresses it. From the resulting spacing (determined by X-ray diffraction) and the directly measured osmotic pressure, we find a force vs. distance curve for compressing the lattice (or, equivalently, the free energy of transfer to bulk water of water between bilayers. This method reveals a very strong, exponentially varying "hydration force" with a decay distance of about 2 A.     

The structural diversity of DNA–neutral phospholipids–divalent metal cations aggregates: a small-angle synchrotron X-ray diffraction study

We investigate the structure of aggregates formed due to DNA interaction with saturated neutral phosphatidylcholines [dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine] in presence of Ca²⁺ and Mg²⁺ cations using simultaneous synchrotron small- and wide-angle X-ray diffractions. For DPPC:DNA = 3:1 mol/base and in the range of 1–50 mM Ca²⁺, the diffractograms show structural heterogeneity of aggregates. We observe the coexistence of two lamellar phases in aggregates prepared at 1 mM Ca²⁺: L^x phase with the DNA strands (of unknown organization) intercalated in water layers between adjacent lipid bilayers and L_DPPC phase of DPPC bilayers without any divalent cations and DNA strands. Aggregates prepared in the range 2–50 mM Ca²⁺ show a condensed gel lamellar phase L_g^c with the lipid bilayer periodicity d ≈ 8.0 nm, and the DNA–DNA interhelical distance d _DNA ≈ 5.1 nm. The increase of temperature induces the decrease in the intensity and the increase in the width of the DNA related peak. In the fluid state, the condensed lamellar phase L_α^c gradually converts into L^x phase. The aggregates do not exhibit rippled P_β phase. The thermal behaviour of aggregates was investigated in the range 20–80°C. Applying heating–cooling cycles, the aggregates converted into energetically more favourable structure: a condensed lamellar phase L^c (or L^x) is preserved or we observe lateral segregation of the DNA strands and metal cations (L^x phase) in coexistence with L_PC phase of pure phospholipids. Dedicated to Prof. Dr Klaus Arnold on the occasion of his 65th birthday.     

On the localization of ubiquinone in phosphatidylcholine bilayers

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05–0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [¹³C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the ¹³C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (Pβ′) phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.     

Study of the orientational ordering of carotenoids in lipid bilayers by resonance-Raman spectroscopy

The orientational ordering of beta-carotene and crocetin embedded in lamellar model membranes has been investigated by angle-resolved resonance Raman scattering at a temperature well above the phase transition of the lipid chains. It is shown that the ordering of the carotenoids is dependent on the chemical composition of the lipid bilayers. The orientational distribution functions found clearly show that beta-carotene is oriented parallel to the bilayer plane (dioleoyl lecithin) or perpendicular to it (soybean lecithin). For dimyristoyl lecithin at 40 degrees C, egg-lecithin, and digalactosyl diacylglycerol two maxima were found in the orientational distribution: one parallel and one perpendicular to the bilayer surface. Crocetin embedded in soybean lecithin bilayers yields a similar bimodal distribution function. Because of rapid photodegradation no results could be obtained for spirilloxanthin.     

Morphology of lipid micelles containing lysolecithin.

Mixtures of lysolecithin with various phospholipids were studied by electron microscopy using negative staining. Mixtures of dipalmitoyllecithin and lysolecithin produced disc-shaped structures which were stacked in aggregates with a 6.0--6.4 nm repeat. The disc were 10--50 nm in diameter. The disc-shaped structures were best observed in equimolar mixtures of dipalmitoyllecithin and lysolecithin. When dipalmitoyllecithin was replaced by dimyristoyllecithin, the structures were rather different from those observed in the system containing dipalmitoyllecithin; a cylindrical micellar phase was predominant. Equimolar mixtures of egg lecithin and lysolecithin formed the more usual smectic, concentric lamellae (liposomes) and elongated rod-like micelles which might be bimolecular fragments of spherules. The radius of the rod-like micelles was about 6 nm. Structures of rod-like micelles were observed more frequently in the samples after incubation at room temperature and then further incubation at 0 degrees C. Equimolar mixtures of didecanoyllecithin and lysolecithin produced large amounts of elongated rod-like micelles. Beef brain sphingoymyelin showed disc-shaped structures when mixed with lysolecithin. Incorporation of cholesterol into the mixtures of dipalmitoyllecithin and lysolecithin changed the morphological structure; the size of the disc became larger and eventually liposomes were formed with an increase of cholesterol content. The structures observed in mixtures of dipalmitoyllecithin or sphingomyelin and lysolecithin closely resembled those observed in complexes of apolipoprotein and lipid.     

Anisotropic and restricted molecular motion in lecithin bilayers

A treatment has been developed which accounts for the prominent features of the PMR spectra and proton relaxation data of unsonicated lecithin bilayers. The spin-lattice relaxation time T₁ and the spin-spin relaxation time T₂ of the protons of a methyl top undergoing anisotropic and restricted reorientation have been calculated employing the theory of Woessner, but modified to include the effects of orientational anisotropies on long time scales. Analysis of the nmr data in terms of this theory permitted determination of the mobility of the lecithin hydrocarbon chains in the bilayer phase. These results indicate that the hydrophobic core of an unsonicated bilayer is more appropriately described as a soft solid rather than as a highly mobile fluid, contrary to the suggestions of recent esr spin label studies.