1. The surface behaviour of six different gangliosides and eight chemically related glycosphingolipids was investigated in monolayers at the air-water interface. 2. Mono-, di-, tri and tetra-hexosylceramides had force-area isotherms showing similar limiting molecular areas on 145 mM-NaCl, pH 5.6. The increasing number of negatively charged sialosyl residues in mono-, di- and tri-sialogangliosides induced a progressive increase in the liquid-expanded character of the films and in the limiting area occupied per molecule, owing to electrostatic repulsions. When the ganglioside monolayers were spread on subphases at pH 1.2, the limiting area per molecule was similar to that found for neutral glycosphingolipids. 3. The monolayer collapse pressure at pH 5.6 increased with the number of uncharged carbohydrate units up to when the polar head group contained 3-4 residues. For gangliosides the collapse pressures were lower and decreased from mono- to tri-sialogangliosides. Ganglioside monolayers on subphases at pH 1.2 showed increases in their collapse pressure. 4. The glycosphingolipid monolayers studied had various surface in their collapse pressure. 4. The glycosphingolipid monolayers studied had various surface potentials according to the complexity of the polar head group of the lipid. Attempts to calculate the dipolar contributions to the surface potential from each carbohydrate residue suggest that the second and third sialosyl residues in di- and tri-sialogangliosides contributed with a vertical dipole moment opposite to that of the first sialosyl residue.
Lipid lateral organization is increasingly found to modulate membrane-bound enzymes. We followed in real time the reaction course of sphingomyelin (SM) degradation by Bacillus cereus sphingomyelinase (SMase) of lipid monolayers by epifluorescence microscopy. There is evidence that formation of ceramide (Cer), a lipid second messenger, drives structural reorganization of membrane lipids. Our results provide visual evidence that SMase activity initially alters surface topography by inducing phase separation into condensed (Cer-enriched) and expanded (SM-enriched) domains. The Cer-enriched phase grows steadily as the reaction proceeds at a constant rate. The surface topography derived from the SMase-driven reaction was compared with, and found to differ from, that of premixed SM/Cer monolayers of the same lipid composition, indicating that substantial information content is stored depending on the manner in which the surface was generated. The long-range topographic changes feed back on the kinetics of Smase, and the onset of condensed-phase percolation is temporally correlated with a rapid drop of reaction rate. These observations reveal a bidirectional influence and communication between effects taking place at the local molecular level and the supramolecular organization. The results suggest a novel biocatalytic-topographic mechanism in which a surface enzymatic activity can influence the function of amphitropic proteins important for cell function.
Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM-->Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of two-dimensional electrostatic fields, based upon the specific orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epifluorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be specific for the SMase-driven system since they could not be observed with Cer-enriched domains generated by defined mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (pi = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-specific packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specific out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (microm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.
The thermotropic behavior of 20 chemically related glycosphingolipids (GSLs) of high purity, containing neutral and anionic carbohydrate residues in their oligosaccharide chains, was studied by high-sensitivity differential scanning calorimetry. In general, the polar head group of GSLs appears to be one of the major determinants of their phase behavior. Compared to phospholipids, the presence of the carbohydrate rather than the phosphorylcholine moiety in the polar head group and a sphingosine base in the hydrocarbon portion of GSLs reduces the effect on the transition temperature (Tm) brought about by increasing the number of methylene groups in the amide-linked fatty acyl chains. For simple neutral GSLs, the Tm's were 20-40 degrees C higher than those of phospholipids with comparable hydrocarbon chains. As the oligosaccharide chain of GSLs becomes more complex, the excess heat capacity, Tm, enthalpy (delta Hcal), and entropy of the transition decrease proportionally to the number of carbohydrate residues present in the polar head group. The Tm and delta Hcal for anionic GSLs were 16-25 degrees C and 1-3 kcal mol-1 lower than those of neutral GSLs with comparable oligosaccharide chains. A linear dependence of delta Hcal with Tm was found. However, the slopes of these plots were different for neutral and for anionic GSLs, suggesting different types of intermolecular organizations for the two. The Tm and delta Hcal were linearly dependent on the molecular area of both neutral and anionic GSLs; this indicated that the influence of the complexity of the polar head group in GSLs for establishing the thermodynamic behavior may be mediated by the intermolecular spacings.
Sphingomyelinase activity against pure sphingomyelin monolayers is constant up to a surface pressure of 18 mN/m and falls above it. Sphingomyelinase- and phospholipase A2-mediated phosphohydrolytic pathways are mutually modulated by the presence of their respective substrates and products. At 15 mN/m non-substrate lipids such as ceramide at a mole fraction of 0.1 in mixed films with the pure substrate, inhibit the sphingomyelinase activity. Ganglioside GM1, another ceramide-containing complex sphingolipid, also inhibits sphingomyelinase activity, while a chemically related glycosphingolipid such as asialo-GM1 has no effect. The activity is unaltered by dipalmitoylphosphatidylcholine and by an equimolar mixture of its products of hydrolysis by phospholipase A2, fatty acid and lysoderivative, but it is inhibited by only one of them or by dilauroylphosphatidylcholine. Phospholipase A2 is inhibited by sphingomyelin, and activated by ceramide and by palmitic acid, one of the products of its own phosphohydrolytic reaction.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.