Sphingomyelin Bilayers Research Articles

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 °C) were treated with sphingomyelinase in the temperature range 10–70 °C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 °C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 °C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.

Comparison of Triton X-100 Penetration into Phosphatidylcholine and Sphingomyelin Mono- and Bilayers

This study has addressed the question whether there is a difference in resistance to Triton X-100 penetration of sphingomyelin and phosphatidylcholine membranes. We have studied this by measuring the penetration of Triton X-100 into monolayers of either d-erythro-N-palmitoylsphingomyelin or dipalmitoylphosphatidylcholine (DPPC) at constant lateral surface pressure and 22 °C. In addition, we also compared how Triton X-100 penetrated into egg phosphatidylcholine and d-erythro-24:1Δ15c sphingomyelin monolayers and how the presence of cholesterol affected the penetration process. In bilayer membranes we assessed Triton X-100 insertion and solubilization by studying the thermodynamics of the partition process and determining the detergent:ratio at which the solubilization is initialized. The penetration of Triton X-100 from the subphase into the monolayers was concentration and surface pressure dependent. The penetration increased linearly with time and concentration for DPPC monolayers, but with sphingomyelin monolayers, the response was clearly biphasic and faster. If the penetration was allowed to proceed, the monolayers eventually became unstable and collapsed (at collapse the detergent-to-lipid ratio was about 1:4 in the monolayer). Inclusion of 30 mol % cholesterol in the monolayers made them more resistant to Triton X-100 penetration, irrespective of the host phospholipid type. In bilayers the partition coefficient (K) becomes lower and the enthalpy change (ΔH) higher for partitioning of Triton X-100 into d-erythro-N-palmitoylsphingomyelin as compared to into DPPC. Onset of solubilization was reached at lower detergent:lipid ratios in d-erythro-N-palmitoylsphingomyelin than in DPPC vesicles. In conclusion, we have observed that d-erythro-N-palmitoylsphingomyelin monolayers do not show increased resistance toward Triton X-100 penetration as compared with membranes prepared from an acyl-matched glycerophospholipid, at least not at room temperature and not even when cholesterol is present. Sphingomyelin bilayers are more susceptible to Triton X-100 solubilization than phosphatidylcholine bilayers, although K was lower for Triton X-100 into sphingomyelin bilayers.

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (TM). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher TMs than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.

Hydrophilic bile salts preserve integrity of sphingomyelin (SM) plus phosphatidylcholine (PC) bilayers by enhancing asymmetric distribution of SM and PC between micellar and vesicular phases

Hydrophilic bile salts preserve integrity of sphingomyelin (SM) plus phosphatidylcholine (PC) bilayers by enhancing asymmetric distribution of SM and PC between micellar and vesicular phases

Interaction of Scopolamine and Cholesterol with Sphingomyelin Bilayers by FT-Raman Spectroscopy

The interaction of scopolamine and cholesterol with sphingomyelin bilayers has been investigated by FT-Raman spectroscopy in head-group region (600-1000 cm-1), the C-C stretching (1000-1200 cm-1), CH2 deformation (1400-1500 cm-1) and the C-H stretching (2800-3000 cm-1) mode regions. The results indicate that scopolamine and cholesterol do not change the conformation of O-C-C-N+ backbone in the choline group of sphingomyelin bilayers, the polar headgroup is still extending parallel to the bilayer surface and 0-C-C-N+ group is still in its gauche conformer. Scopolamine and cholesterol lower the order of the interface, the interchain, CH2 crystal lattices and the lateral chain-chain packing, and increase their fluidity.

Lipid domains in the membrane: thermotropic properties of sphingomyelin vesicles containing GM1 ganglioside and cholesterol.

The thermotropic behavior of palmitoylsphingomyelin vesicles containing GM1 ganglioside and cholesterol has been investigated by high-sensitivity differential scanning calorimetry. The thermograms exhibited by binary palmitoylsphingomyelin/GM1 mixtures are resolvable into two components. The relative contribution of the minor component, undetectable in the absence of ganglioside, to the total enthalpy and its transition temperature (>40 degrees C) increase with the concentration of the glycolipid embedded in the vesicles. These data suggest the occurrence of lateral phase separation and that more ordered, higher melting GM1 ganglioside-enriched domains are present within the sphingomyelin bilayer. Studies on binary sphingomyelin/cholesterol mixtures confirmed the known tendency of the sterol to decrease the total enthalpy of sphingomyelin, forming cholesterol-enriched domains. The thermograms exhibited by ternary sphingomyelin/ganglioside/cholesterol mixtures in variable proportions (up to 20% molar GM1 or Chol) displayed, on increasing the content of either the sterol or the ganglioside, features addressable to sphingomyelin/cholesterol (peaks centered at temperature </=40 degrees C, decrease of enthalpy) or to sphingomyelin/GM1 mixtures (peaks centered at a temperature >40 degrees C), respectively. This trend was confirmed by deconvolution analysis, showing that the thermograms are resolvable into components addressable to GM1-enriched and to cholesterol-enriched domains. Taken all together, the results show that the architectural features of sphingomyelin bilayers are strongly dependent on the presence of GM1 ganglioside and cholesterol, whose presence is leading to the formation of separate, GM1-enriched and cholesterol-enriched distinct domains. Ganglioside-sphingomyelin and sphingomyelin-cholesterol, together with mutual ganglioside-ganglioside, interactions could contribute to maintain a network of bonds extending to proteins, forming specialized membrane domains, such as caveolae, or others, whose experimental clues are the glycolipid-enriched detergent-insoluble fractions that can be isolated from cell membranes.

Effect of sphingomyelin composition on the phase structure of phosphatidylcholine-sphingomyelin bilayers.

In this study, we examine the phase behavior as well as lateral diffusion and percolation in the region of coexisting gel and fluid phases in binary mixtures composed of dimyristoylphosphatidylcholine and one of two totally synthetic D-erythro-sphingomyelins (having either C16 or C24 acyl chains, both having similar gel to liquid-crystalline phase transition temperatures). This study stems from the uniqueness of sphingomyelins having gel to liquid-crystalline transition temperatures in the range of physiological interest, and the fact that more than 50% of the naturally occurring sphingomyelin species have a chain mismatch. The presence of sphingomyelin in biological membranes can thus be expected to give rise to a complex phase structure. Fluorescence recovery after photobleaching, differential scanning calorimetry, and electron microscopy are used to show that, despite similarity in the temperature range of the gel to liquid-cystalline phase transition of the two sphingomyelins, the two differ in their phase structure. Also they differ to a large extent in their mixing with dimyristoylphosphatidylcholine. Dimyristoylphosphatidylcholine and C16 sphingomyelin mix nearly ideally, with the percolation threshold locus lying close to the liquidus on the phase diagram. In contrast, the C24 sphingomyelin and dimyristoylphosphatidylcholine mix nonideally, with the percolation threshold locus lying close to the solidus. In addition, mixtures containing C24 sphingomyelin have a complex thermotropic behavior which may be related to the observation that these dispersions contain several types of particles, some of which are not multilamellar vesicles. These studies suggest that the degree of sphingomyelin chain mismatch is an important factor in determining lateral organization in the membrane.

Fluorescence evidence for cholesterol regular distribution in phosphatidylcholine and in sphingomyelin lipid bilayers

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.

Topography and thermotropic properties of cannabinoids in brain sphingomyelin bilayers.

In our previous publications we compared the locations of the biologically active (-)-delta 8-tetrahydrocannabinol (delta 8-THC) with that of its inactive analog O-methyl-(-)-delta 8-tetrahydrocannabinol (Me-delta 8-THC) in the liquid crystalline phase of partially hydrated dimyristoylphosphatidylcholine (DMPC) bilayers (Mavromoustakos et al. (1990) Biophys. Acta 1024, 336-344; Yang et al. (1993) Life Sci. 53, 117-122). delta 8-THC was shown to localize itself preferentially in the vicinity of the membrane interface with its phenolic hydroxyl group anchored near the carbonyl groups of DMPC while the more lipophilic Me-delta 8-THC is located deeper towards the center of the bilayer. In the present publication we studied and compared the topography of the two analogs in the gel phase of brain sphingomyelin bilayers. Again we found that delta 8-THC is located near the membrane interface approximately 15 A from the center of the bilayer while its inactive analog localizes deeper in the bilayer at an average site only 8 A from the center of the membrane bilayer. It thus, appears that both analogs preferentially localize in distinct sites within the membrane bilayer which are independent of the mesomorphic state and the nature of the phospholipid. Our results suggest that in the more complex environment of biological membrane which is composed of different phospholipids and proteins the two analogs are expected to prefer different average locations within the bilayer, a property which may in part explain the observed differences in their biological activities.

NMR and FT-Raman Studies on the Interaction of Lanthanide Ions with Sphingomyelin Bilayers

Abstract The interactions of lanthanide ions with sphingomyelin bilayers have been studied by using 2D NOESY spectroscopy and FT-Raman spectroscopy methods. The results indicate that lanthanide ions, as well as divalent calcium, combine mainly to the phosphate group in the polar headgroup and do not change the conformation of O-C-C-N+ backbone in the choline group of sphingomyelin bilayers. The polar headgroup is still extending parallel to the bilayer surface and O-C-C-N+ group is still in its gauche conformer.

Incorporation of proteins in sphingomyelin-water gel phases

Sphingomyelin from bovine milk and water form lipid gel phases at room temperature. A sample was used which incorporated of about 55% water, and X-ray diffraction data indicate an aqueous layer thickness of about 28 Å. In order to accommodate proteins in the gel phase, the aqueous layer thickness was increased by solubilizing sodium palmitate into the sphingomyelin bilayer. In this way the gel phase could take up about 80% water. The incorporation of lysozyme, β-lactolylobulin, and α-lactalbumin, was followed and the protein concentration for phase separation to occur was determined. It was found that the degree of incorporation was dependent on the salt concentration, thus the protein used was extensively dialysed. The amount of protein which can be dissolved in the 'thin aqueous layer of the gel phase was suggested to be limited by the dimensions of the layer. These are likely to be reduced as a consequence of the osmotic stress exerted by the 'outside' solution phase at high enough protein concentration.

N-palmitoyl sphingomyelin bilayers: structure and interactions with cholesterol and dipalmitoylphosphatidylcholine.

The structure and thermotropic properties of N-palmitoyl sphingomyelin (C16:0-SM) and its interaction with cholesterol and dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry (DSC) and X-ray diffraction methods. DSC of hydrated multi-bilayers of C16:0-SM shows reversible chain-melting transitions. On heating, anhydrous C16:0-SM exhibits an endothermic transition at 75 degrees C (delta H = 4.0 kcal/mol). Increasing hydration progressively lowers the transition temperature (TM) and increases the transition enthalpy (delta H), until limiting values (TM = 41 degrees C, delta H = 7.5 kcal/mol) are observed for hydration values > 25 wt % H2O. X-ray diffraction at temperatures below (29 degrees C) TM show a bilayer gel structure (d = 73.5 A, sharp 4.2 A reflection) for C16:0-SM at full hydration; above TM, at 55 degrees C, a bilayer liquid-crystal phase is present (d = 66.6 A, diffuse 4.6 A reflection). Addition of cholesterol to C16:0-SM bilayers results in a progressive decrease in the enthalpy of the transition at 41 degrees C, and no cooperative transition is detected at > 50 mol % cholesterol. X-ray diffraction shows no difference in the bilayer periodicity, position/width of the wide-angle reflections, or electron density profiles at 29 and 55 degrees C when 50 mol % cholesterol is present. Thus, cholesterol inserts into C16:0-SM bilayers progressively removing the chain-melting transition and changing the structural characteristics of the bilayer. DSC and X-ray diffraction data show that DPPC is completely miscible with C16:0-SM bilayers in both the gel and liquid-crystalline phases; however, 30 mol % C16:0-SM removes the pre-transition exhibited by DPPC.

Structure and molecular conformation of anhydrous and of aqueous sphingomyelin bilayers determined by infrared and Raman spectroscopy

Fourier transform-infrared spectra of anhydrous sphingomyelin and sphingomyelin bilayers in aqueous and deuterium phases have been investigated in detail. Raman spectra of the aqueous gel have also been measured. From these vibrational data, the structure and the molecular conformation of the head group, interface region and the hydrocarbon chains have been deduced in the solid and bilayer phases of the lipid. Vibrational assignments for the prominent fundamental modes associated with secondary structure of the lipid and molecular conformation of sphingosine and acyl chains, peptide unit and phospho-choline moiety are proposed. Vibrationally complex 0H and NH stretching and deformation regions of the peptide unit and of bilayer water molecules have been probed using isotopic deuterium substitution. Infrared data suggest a strong in-termolecular hydrogen bonding for the 3-hydroxyl groups of the sphingosine chains and an unusual intramolecular hydrogen bonding for the amide protons to carbonyl oxygens in the bilayer. These hydrogen-bonding schemes are likely to increase membrane stability through lateral contacts which may play an important role in membrane regulatory functions. Second derivative infrared spectroscopy has been used to explore the structurally sensitive spectral regions such as carbonyl, methylene deformation and phosphate regions of the lipid. This application revealed many weak bands associated with the specific regions of the membrane structure. Temperature-induced cooperative conformational transitions in sphingomyelin biomembrane have been examined in the aqueous phase. Data suggest a two state order-disorder phase transition at 35°C with a transition enthalphy of 6.5 kcal mol −1, agreeing closely with the enthalpies derived for structurally similar systems. The hydrocarbon chains are packed in a pseudo- or distorted hexagonal lattice in the solid and the gel phases. Data also indicate the existence of a hybrid domain in which lipid molecules coexist over a wide temperature range in the vicinity of the transition temperature, but revealed no evidence of interdigitation of hydrocarbon chains proposed earlier. Raman data also suggest that at least 30% of the hydrocarbon chain segments adopt a gauche conformation in the gel phase as well as in the anhydrous solid at room temperature. This indicates a considerable non-rigid environment for the sphingolipid hydrocarbon chains in the anhydrous and gel phases.

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.

Structure and thermotropic properties of hydrated N-stearoyl sphingomyelin bilayer membranes

Hydrated multibilayers of N-stearoyl sphingomyelin were investigated as a function of hydration using differential scanning calorimetry (DSC) and X-ray diffraction. Anhydrous N-stearoyl sphingomyelin exhibits an endothermic transition at 75°C (Δ H=3.8kcal/mol) are increasing hydration proessively lowers the transition temperature and increases the transition enthalpy, until limiting values ( T m = 45°C, Δ H = 6.7kcal/mol) are observed for hydration values > 21.4% H 2O. At low hydration levels, < 20% H 2O, an additional transition is observed at approx. 20°C. X-ray diffraction studies at temperatures below (22°C) and above (55°C) the main endothermic transition confirm that the bilayer gel (sharp 4.2Åreflection) → bilayer liquid crystal (diffuse 4.5Åreflection) transition occurs at all hydration levels with limiting bilayer hydration occurring at approx. 31.5% H 2O in the gel phase and at approx. 35% H 2O in the liquid crystal phase. The thermotropic properties and metastability of this partial synthetic N-stearoyl sphingomyelin differ in some respects from that of the previously studied synthetic dl-erythro- N-stearoyl sphingomyelin (Estep, T.N., Calhoun, W.I., Barenholz, Y., Biltonen, R.L., Shipley, G.G. and Thompson, T.E. (1980) Biochemistry 19, 20–24), suggesting an influential role of the interfacial molecular conformation.

Nuclear magnetic resonance study of sphingomyelin bilayers

Bilayers of D-erthro-(N-stearoylsphingosyl)-1-phosphocholine (C18-SPM), previously characterized by differential scanning calorimetry [Bruzik, K. S., & Tsai, M.-D. (1987) Biochemistry 26, 5364-5368] in various phases, were studied by means of wide-line 31P, 2H, high-resolution 13C CP-MAS, and 1H MAS NMR. The fully relaxed gel phase of C18-SPM at temperatures below 306 K displayed 31P NMR spectra characteristic of the rigid phase with frozen rotation of the phosphocholine head group. Three other gel phases existing in the temperature range 306-318 K displayed spectra with incompletely averaged axially symmetric powder line shapes and were difficult to differentiate on the basis of their 31P NMR spectra. The gel-to-gel transition at 306 K was found to be fully reversible. The main phase transition at 318 K resulted in the formation of the liquid-crystalline phase for which spectra with axially symmetric line shapes of uniform width were obtained, regardless of the nature of the starting gel phase. 13C CP-MAS NMR spectra revealed significant differences in the molecular dynamics of sphingomyelin in various phases. All carbon atoms of the polar head group in the liquid-crystalline phase gave rise to a separate resonance lines. Numerous carbon atom signals were doubled in the stable phase, demonstrating the existence of two slowly interconverting conformers.

Interaction of furosemide with lipid membranes.

The interaction of furosemide with different phospholipids was investigated. Its influence on the lipid structure was inferred from its effect on the phase transition properties of lipids and on the conductance of planar bilayer membranes. The thermotropic properties of dipalmitoyl phosphatidylcholine, phosphatidylethanolamine (natural), dipalmitoyl phosphatidylethanolamine, brain sphingomyelin, brain cerebrosides and phosphatidylserine in the presence and absence of furosemide were investigated by differential scanning calorimetry. The modifying effect of furosemide seems to be strongest on phosphatidylethanolamine (natural) and sphingomyelin bilayers. The propensity of furosemide to decrease the electrical resistance of planar lipid membranes was also studied and it is shown that the drug facilitates the transport of ions. Partition coefficients of furosemide between lipid bilayers and water were measured.

Interactive forces between sphingomyelin bilayers

The net repulsive force as a function of bilayer separation was measured for sphingomyelin in water and in 30 m M CaCl 2. The decay of the hydration repulsive force, 6.27 ± 0.23 Å, and the decay of the repulsive electrostatic force, 16 ± 1 Å, for sphingomyelin bilayers were found to be consistent with force decays previously observed for phosphatidylcholine bilayers. Bilayer interactive forces appear to be somewhat influenced by changing the backbone moiety of the choline-containing lipid from glycerol to sphingosine. The influence of just the lipid head groups on the van der Waals attractive force was less clear since the van der Waals force is a result of influences from both the head group and acyl chain regions of the lipid in addition to the aqueous environment.

Characterization of the association of Electrophorus electricus acetylcholinesterase with sphingomyelin liposomes. Relevance to collagen-sphingomyelin interactions

Electrophorus electricus acetylcholinesterase is a large polymorphic enzyme. Its native forms 18 S, 14 S and 8.5 S posses a tail having a collagen-like structure. It was suggested that this tail is involved in the anchorage of the enzyme at the terminal of the synapse. Watkins et al. [1] showed that all forms of the enzyme having a collagen segment also bind sphingomyelin liposomes with almost no binding to phosphatidylcholine (PC) liposomes. In agreement with the above results, the binding of acetylcholinesterase reported here was independent of the following liposomal parameters (a) curvature, (b) the physical state of the bilayer, (c) the gel to liquid crystalline phase transition of sphingomyelin, (d) stereospecificity of the sphingomyelin, (e) acyl chain of the sphingomyelin. The binding was reduced with increasing PC content in sphingomyelin vesicles. The binding has no effect on the bilayer integrity. The enzymatic activity can be released from the vesicles by incubation with collagenase. The association of the enzyme with the liposomes had minimal effect on its kinetic parameters ( K m, V max). The only detectable effect was increasing enzyme stability at low enzyme concentration. This suggested that the binding of the enzyme to sphingomyelin liposomes reduced its surface denaturation. Such association was not unique to acetylcholinesterase since collagen showed similar behavior. Collagen binding to sphingomyelin liposomes was 5–10 times larger than to PC liposomes. The exact details of the interaction of collagen and collagen-like peptides with sphingomyelin bilayers are yet unknown although it differs from the well documented hydrophobic or electrostatic interactions [7]. This work proposes hydrogen bonding as a third mechanism which involves the interface region of sphingolipids molecules and the collagen or collagen-like tail of acetylcholinesterase. This binding is also of interest due to its correlation to the accumulation of sphingomyelin and collagen during aging and the development of atherosclerosis in blood vessels of mammals.

Direct determination by Raman scattering of the conformation of the choline group in phospholipid bilayers.

For clarification of the assignment of the vibrational modes of the choline group, Raman spectra of choline iodides selectively deuterated at three different positions were investigated. The isotope shifts of the C-N stretching vibrations suggested that they are conformation sensitive. When the Raman spectra of choline chloride, carbamoylcholine iodide, carbamoylcholine chloride, and methoxycarbonylcholine iodide are compared with the crystal structures of these compounds, a correlation between the vibrational frequency and the conformation of the O-C-C-N+ backbone could be established. The Raman bands attributed to the "totally" symmetric stretching (v1) and symmetric stretching vibrations (v2) of the C-N bonds of the quaternary ammonium group appeared at about 720 cm-1 and about 870 cm-1, respectively, for the gauche conformation of the O-C-C-N+ backbone, and in the trans conformation, they shifted to about 770 cm-1 (v1) and about 910 cm-1 (v2), respectively. On the basis of this correlation and from measurements of phosphatidylcholine and sphingomyelin bilayers, it was concluded that most of the choline groups in both bilayers take the gauche conformation not only in the solid state but also in the gel and liquid-crystalline states. These data represent the first direct evidence that a gauche conformation for the O-C-C-N+ bond is preferred in the gel and liquid-crystalline states. These key bands, especially the v1 band, are a powerful tool to study the conformation of the choline group in situ not only in the membrane field but also in the neuroscience in connection with acetylcholine.