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The present paper reports the experimental observation of an interesting thermodynamic process which could be biologically important: such behaviour, shown by some gangliosides, is likely to be peculiar of these glycosidic compounds as it has never been observed for other membrane-type amphiphilic molecules. In water solution, gangliosides have been found to present a bistable behaviour between two stable states (called A and B) which does not involve any change in the primary structure of the molecule. The interconversion between state A and state B, and vice versa, does not occur spontaneously, but has to be triggered by some external agent, which makes this system a potentially regulated process with important biological correlations. In the present experiments, state B is reached from state A with a temperature rise in the range 30-55 degrees C. The new state is stable regardless of any possible temperature cycle. The initial state A is then regained when the ganglioside solution is dried and the solute is redissolved. The two states are believed to correspond to two different conformations of the hydrophilic portion of the molecule. The bistable behaviour is shown by the gangliosides GM2, GM1, GD1a, GD1b and Fuc-GD1b, GT1b, however, does not show such an effect.

GM1 ganglioside containing a hydroxylated fatty acid moiety, GM1(OH), was synthesized starting from lyso-GM1 and D-(+)-2-hydroxystearic acid. The aggregative, geometrical and distribution properties of GM1(OH) were compared with those of stearic acid containing GM1 ganglioside; laser light scattering measurements, differential scanning calorimetry and fluorescence spectroscopy were used. GM1 and GM1(OH) are present in solution as micelles with a hydrodynamic radius of 58.7 and 60.0 A, and molecular mass of 470 and 570 kDa, respectively. The surface area occupied by the monomer of GM1(OH) at the lipid-water interface of the aggregate was calculated to be 117 A2, which is 3 A2 lower than that determined for GM1. Proton NMR analyses of GM1 and GM1(OH) suggest different three-dimensional structures at the ganglioside lipid-water interface. Both GM1(OH) and GM1 inserted into dipalmitoylphosphatidylcholine (DPPC) vesicles undergo segregation phenomena, with the formation of ganglioside-enriched microdomains, but GM1(OH) shows a higher degree of dispersion in the DPPC matrix and exerts a lower rigidifying effect than does GM1.

A study has been made of the association properties of the two GM1 ganglioside molecular species GM1-C18 and GM1-C20 (containing C18 and C20 long chain bases, respectively) to rat cerebellar granule cells in culture. Both gangliosides recognized, to the same extent, and associated with them to give a form of association, the trypsin-labile form. This form was removed by treatment with trypsin enzyme. Both gangliosides associated stably with the cells to become components of the cell membranes. Although similar amounts of the two gangliosides entered the cells, being then metabolized, the time course of the association was different for the two gangliosides: after 15 h of ganglioside-cell incubation the amount of GM1-C18 inserted into the cell membrane was 2.43 times higher than that of GM1-C20.

The aggregative properties of GM1 ganglioside containing an acetyl group as acyl moiety [GM1(acetyl)] in aqueous solution have been studied by static and dynamic light scattering measurements and surface tension experiments. GM1 (acetyl) spontaneously aggregates as small micelles showing a hydrodynamic radius and molecular weight of 34 A and 102 kDa, respectively, down to a concentration of 2.0 x 10(-5) M.

A light scattering study of the effect of mixing in aqueous solution two gangliosides, GM2 and GT1b, having different hydrophilic headgroups and similar lipid moieties is presented. Mixed micelle formation with spatial segregation of one ganglioside with respect to the other was observed. It is also shown that segregation is a spontaneous phenomenon which is explainable only in terms of simple geometrical arguments, that is by the fact that the large headgroup of GT1b provides the lipidic core of the aggregate with a better shielding from water in the highly curved regions than the smaller headgroup of GM2 can do. This finding may be of help in understanding the behaviour of gangliosides in artificial and natural membranes.

The aggregative properties of highly pure GM3 ganglioside in aqueous solution have been studied by static and dynamic laser light scattering measurements and fluorescence spectroscopy experiments performed by the use of a GM3 derivative containing the pyrene group at the end of the fatty acid moiety. GM3 ganglioside spontaneously aggregates as unilamellar vesicles, down to a concentration of 1.25 x 10(-8) M, showing molecular weight and hydrodynamic radius ranging from 15,000 to 30,000 kDa and from 350 to 470 A, respectively. GM3 vesicles are stable with dilution and can be stored at room temperature for some weeks without appreciable change.

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.

Static and dynamic laser light scattering measurements on micellar aqueous solutions of gangliosides GM2, GM1, GD1a are reported. The aggregation number, the hydrodynamic radius and the micellar shape depend on the type of ganglioside and the unsaturation degree of the hydrocarbon chains. At a temperature of 25 degrees C the molecular weights of GM2, GM1 and GD1a are 740,000, 470,000 and 418,000 DA respectively. A significant decrease of micellar size with temperature has been found for saturated GM1 in the region 25 degrees-40 degrees C. The strong sensitivity of the micellar parameters to the ganglioside structure is explained by making reference to some simple model which takes into account geometrical packing considerations. By measuring the scattered light intensity at low ionic strength of the solution (0.1-30 mE) it was possible to evaluate the ganglioside micellar charge, 100 electronic units for GM2, 48 for GM1 and 60 for GD1a.

A new chemical procedure is described for preparing labelled GM1 molecular species, carrying as acyl moiety pyrene-decanoic acid, 5-doxyl-stearic acid and 16-doxyl-stearic acid. It makes use of a mixed anhydride formed by ethylchloroformate and the labelled acyl chain, as the reagent for N-acylation of a deacetylated, deacylated GM1 ganglioside, which is prepared by alkaline hydrolysis of natural GM1. The reaction performed with a unitary GM1 derivative/mixed anhydride molar ratio, occurs with a yield of above 40%. The labelled deacetylated GM1 molecular species are then N-acetylated by means of acetic anhydride with quantitative yield. The chemical process of insertion of labelled fatty acid and reconstitution of GM1 ganglioside has been confirmed by GLC-MS and NMR analyses. Fluorescence and electron spin resonance experiments indicate that the labelled gangliosides behave similarly to natural GM1, in both the aggregation properties and the capability to be transferred from micelles to vesicular dispersions of phospholipids.

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 MM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15-37 degrees C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.

N-Glycolylneuraminic acid containing GM1, GM1(NeuGc), was prepared by semisynthetic procedure. The procedure makes use of GM1 ganglioside deacetylated at the level of sialic acid residue (deAc-GM1) and of 1,3-dioxalan-2,4-dione. DeAc-GM1 is prepared from GM1 by alkaline hydrolysis in the presence of tetramethylammonium hydroxide and the glycolylating compound by reaction of glycolic acid with phosgene in dioxane, followed by cyclization under vacuum. Mass spectrometric and nuclear magnetic resonance spectroscopy analyses clearly indicated the presence, in the neosynthesized ganglioside of a glycolic group in the sialic acid residue. Laser-light scattering measurements show that GM1(NeuGc) aggregates in aqueous media being present in solution as micelles with a molecular weight of 576,000 and a hydrodynamic radius of 62.4 A as determined at 25 degrees C. GM1(NeuGc) promotes neurite outgrowth in N-2a cells to a similar degree as GM1(NeuAc), but shows different behaviour under treatment with sialidase from Arthrobacter ureafaciens.

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. GM1 and GD1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris--HCl buffer (pH 6.8) at 37 degrees C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 A argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c0) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius RH, and also contained information on the polydispersity of the sample. We find co less than 1 x 10(-6) M for both GM1 and GD1a, M = 532000 +/- 50000 and RH = 63.9 +/- 2 A for GM1, and M = 417000 +/- 40000 and RH = 59.5 +/- 2 A for GD1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range, and, in particular of Ca2+, did not influence significantly the values of co and the main features of the micelle.