Two gel states of cerebrosides. Calorimetric and Raman spectroscopic evidence.

The enthalpy of the gel-to-liquid crystalline phase transition for kerasin (15.8 kcal/mol) is found to be markedly higher than that for phrenosin and unfractionated bovine brain cerebrosides (about 7 kcal/mol). Evidence for a higher degree of order in the hydrocarbon chains and a different configuration in the polar region of kerasin is supplied by Raman spectroscopic parameters for these gel phases. The high transition enthalpy for kerasin is ascribed to a lesser accommodation of gauche conformers in the hydrocarbon chains just below the transition temperature. The thermodynamic behavior of these cerebroside fractions, including hysteresis in kerasin gels, is compared to that previously reported for sphingomyelins.

Phase behavior of cerebroside and its fractions with phosphatidylcholines: calorimetric studies.

Bovine brain cerebroside and its kerasin (beta-D-galactosyl-N-acyl-D-sphingosine) and phrenosin (beta-D-galactosyl-N-(2-D-hydroxyacyl)-D-sphingosine) fractions were mixed with diacylphosphatidylcholines (PCs) to form fully hydrated lamellar phases. These mixtures were examined by differential scanning calorimetry, and phase diagrams for cerebroside/diacylPC mixtures were constructed from the data. Cerebroside was found to be miscible with egg PC at low mole fractions X of cerebroside; the mixture behaves non-ideally for X greater than 0.25. The non-ideal behavior appears to be a superposition of separate interactions of kerasin and phrenosin with egg PC. Strikingly, phrenosin mixes nearly ideally with egg PC. Kerasin mixed with egg PC yields a peritectic phase diagram. Cerebroside and phrenosin were found to be immiscible with dimyristoylphosphatidylcholine (DMPC) in the gel state in low proportions. Both stable and metastable gel phases of kerasin were detected in different endotherms of kerasin/PC mixtures. Kerasin in the stable and metastable gel states exhibits discontinuous and continuous ranges of miscibility, respectively, with DMPC. The stable gel phase of kerasin does not segregate in natural cerebroside. Natural kerasin was found to act isomorphic to semi-synthetic (natural configuration) D-kerasins but not completely to synthetic DL-kerasins of single acyl chain lengths.

Raman spectra and vibrational assignments for deuterated membrane lipids. 1,2-Dipalmitoyl phosphatidylcholine-d9 and -d62.

Vibrational Raman spectra of polycrystalline 1,2-dipalmitoyl phosphatidylcholine-d9 (fully deuterated choline methyl groups) and 1,2-dipalmitoyl phosphatidylcholine-d62 (fully deuterated acyl chains) were recorded in the 3050- 2800, 2250-2050 and 1800-700 cm-1 regions. The fundamental vibrational modes were assigned primarily on the basis of isotopic frequency shift ratios, group frequency correlations and comparisons with specific model compounds. Since deuterium-substituted lipids provide well-isolated spectral probes, particularly in the carbon-deuterium stretching region, the dependence of the 2250-2050 cm-1 region on lipid phase was examined for the dipalmitoyl phosphatidylcholine-d62 species. The methylene CD2 deformation and twisting modes at 984 and 919 cm-1, respectively, also exhibit intense, isolated vibrational transitions which should prove useful for monitoring molecular order in mixed dueterated and undeuterated lipid systems. Except for the relatively weak choline methyl C-D and C-H stretching modes, the spectrum of 1,2-dipalmitoyl phosphatidylcholine-d9 is not distinguishable from that of the undeuterated system. For both the d9 and undeuterated species, the vibrational modes associated with the lipid head group region are sensitive to slight hydration.

Vibrational Raman spectra of lipid systems containing amphotericin B.

Resonance-enhanced and normal vibrational Raman spectra were observed for both multilamellar and single-wall vesicle assemblies of dimyristoyl phosphatidylcholine containing amphotericin B, a channel-forming polyene antibiotic, and cholesterol. The decrease in the frequency of the polyene antibiotic C = C stretching mode at 1556 cm-1 and the increase in intensity of the C-C-H in-plane deformation mode at 1002 cm-1 indicate that amphotericin B is ordered in a lipid-cholesterol medium similarly to the solid, but is surrounded by a slightly more polar environment. The intensity of the C = C stretching mode I1556 decreases 4-fold during the broadened gel to liquid crystalline phase transition (16--32 degrees C) of dimyristoyl lecithin-cholesterol (4 : 1) multilayers. Other resonance-enhanced vibrations of amphotericin B exhibit similar behavior. For amphotericin B in pure dimyristoyl lecithin multilayer or vesicle systems, however, the vibrational intensity associated with the C = C stretching mode remains constant during the melting of lipid hydrocarbon chains. In addition, a third effect occurs in liquid crystalline egg lecithin-cholesterol (4 : 1, mol ratio) multilayers in which I 1556 first increases by 25% between 3 and 25 degrees C, in parallel with the loss of active channels, and then remains constant as the temperature increases from 25 to 42 degrees C. This latter intensity pattern is masked in the dimyristoyl lecithin-cholesterol system by the overwhelming effect upon the C = C mode from changes in the lipid chain packing characteristics which occur during the phase transition. The broadened phase transition in 4 : 1 dimyristoyl lecithin-cholesterol multilayers (16--32 degrees C), as followed by the ratio of intensities at 2880 and 2850 cm-1 (asymmetric and symmetric methylene C-H stretching modes, respectively) is slightly narrowed by the addition of amphotericin B, and effect from which a binding stoichiometry at 24 degrees of 1 : 1 amphotericin B: cholesterol is estimated. This stoichiometry was confirmed by differential calorimetric scans, which also show the presence of a peak proportional to cholesterol content. Raman I2880/2850 peak height ratios in pure dimyristoyl lecithin bilayers were increased over the 14--38 degrees C range by amphotericin B, a spectral effect which suggests an ordering of the lipid matrix perhaps as a consequence of the polyene binding to the bilayer surface. For bilayers containing cholesterol, the ratios of intensities of the 2935 cm-1 feature, composed mainly of acyl chain terminal methyl and underlying methylene C-H stretching modes, to the 2850 cm-1 feature are significantly increased by amphotericin B. This effect indicates that the antibiotic penetrates the bilayer in the lipid-sterol system.

Comment on the carbon-hydrogen stretching region of vibrational Raman spectra of phospholipids.

The broad, environmentally sensitive 2935-2910 cm-1 feature in the vibrational Raman spectra of phospholipids originates primarily from methylene symmetric carbon-hydrogen (C-H) stretching modes. For ordered acyl chains exhibiting local C2h symmetry at low temperatures, the infrared active methylene C-H asymmetric stretching modes become increasingly apparent at higher temperatures in the Raman spectrum as intramolecular chain disorder (trans-gauche isomerization) leads to a loss of chain symmetry. The 2935 cm-1 shoulder, assigned to the acyl chain methyl group C-H symmetric stretching vibration, is difficult to distinguish as a separate spectral transition in the chain disordered state. An ordering of lipid fluidity for several phospholipids is presented in terms of the intensities of features in the 3000-2800 cm-1 region with particular emphasis upon the 2935-2910 cm-1 contour.

Spectrophotometric determination of intracellular pH with cultured rat liver epithelial cells.

A spectrophotometric method using 6-carboxyfluorescein (CF) was developed to determine intracellular pH in anchorage-dependent monolayers of control cells of rat hepatic origin. Until now, such studies have been carried out with ascites cells in suspension, which lack specific controls for comparative studies. The rat cell line is grown on plastic Leighton tube slides which fit directly into 3 cm spectrophotometer cuvettes. One sample, without CF, serves as a control for the light-scattering properties of the cell monolayers. Steady-state determinations show a decline in intracellular pH from 7.3 to 6.8 ten minutes after the addition of glucose and quercetin. Kinetic determinations show that with the addition of glucose to substrate-free cells the rate of acid formation is -0.02 pH units/min; the addition of quercetin results in a further acceleration of the kinetic rate to -0.10 pH units/min. In both types of analyses, the change in intracellular pH is standardized with nigericin and external buffers, based on the decrease in the maximum absorption of CF at 492 nm. The results demonstrate that even with anchorage-dependent monolayers of a control hepatocyte line which produces very little acid, this spectrophotometric method permits determinations sufficiently sensitive for analysis of intracellular pH.

Phase behavior of ganglioside-lecithin mixtures. Relation to dispersion of gangliosides in membranes.

Ganglioside GM1 and mixed brain gangliosides were mixed with 1-stearoyl-2-oleoyl lecithin (SOPC) and examined by differential scanning calorimetry as a function of ganglioside content and temperature. Low mole fractions of ganglioside GM1 and of mixed brain gangliosides are shown to be miscible with SOPC in the gel phase up to X = 0.3, with the possible exception of a small region of immiscibility for the mixed brain gangliosides system centered around X = 0.05. Above X = 0.3, the low-temperature phases demix into a (gel) phase of composition X = 0.3 and a (micellar) phase of composition X = 1.0. Above the endothermic phase transition temperature, no phase boundaries are discerned. It is pointed out that phase structures need to be determined in each domain delineated in the phase diagrams, and that cylindrical phases may exist at higher temperatures and intermediate compositions. The effects of addition of wheat germ agglutinin, which binds to ganglioside GM1, on a ganglioside GM1-SOPC mixture (X = 0.5), are described and interpreted in terms of partial demixing of ganglioside and lecithin. Behavior of the ganglioside-SOPC system is discussed with respect to the kinetics of cholera toxin action in lymphocytes, as well as to other physiological roles of gangliosides in membranes.

Molecular conformations of cerebrosides in bilayers determined by Raman spectroscopy.

Vibrational Raman spectra of the solid and gel phases of bovine brain cerebrosides and the component fractions, kerasin and phrenosin, provide conformational information for these glycosphingolipids in bilayer systems. The carbon-carbon stretching mode profiles (1,150-1,000 cm-1) indicate that at 22 degrees C the alkyl chains assume an almost all-trans arrangement. These spectral data, combined with those from the C-H stretching region (3,050-2,800 cm-1), show that phrenosin forms the most highly ordered polycrystalline solid and kerasin the most ordered gel phase. The conformation of the unsaturated, 24-carbon acyl chains is monitored independently by a skeletal stretching mode at 1,112 cm-1. The alkyl chains in the kerasin and phrenosin gels are sufficiently extended to allow interdigitation of the 24-carbon acyl chains across the midplane of the bilayer. The amide I vibrational mode occurs at a lower frequency in solid phrenosin than kerasin, a shift consistent with stronger hydrogen bounding. This band is broadened and shifted to higher frequencies, however, in the phrenosin gel phase. In both the solid and gel phases natural cerebroside exhibits a composite amide I mode. The disruptive effects on cerebroside chain packing and headgroup orientation arising from mixing with dimyristoyl phosphatidylcholine are examined. Vibrational data for cerebroside are also compared to those for ceramide, sphingosine, and distearoyl phosphatidylcholine structures. Spectral interpretations are discussed in terms of calorimetric and X-ray structural data.