EFFECT OF GLYCOSPHINGOLIPID FATTY-ACID CHAIN-LENGTH ON BEHAVIOR IN UNSATURATED PHOSPHATIDYLCHOLINE BILAYERS - A H-2 NMR-STUDY

Deuterium-labeled glycosphingolipids, N-lignoceroyl[d47]galactosylceramide (24:0 fatty acid form) and N-stearoyl[d35]galactosylceramide (18:0 fatty acid form) were prepared by partial synthesis. These probe-labeled species, differing only in the lengths of their fatty acids, were compared via H-2 NMR with regard to arrangement and behavior in bilayers of the monounsaturated phospholipid 1-stearoyl-2-oleoylphosphatidylcholine (SOPC). Results were used to consider the physical significance of the great range of common acyl chain lengths that is a frequent feature of cell membrane glycosphingolipids. N-lignoceroyl[d47]- and N-stearoyl[d35]galactosylceramide were incorporated at concentrations ranging from 5 to 50 mol % into unsonicated phospholipid liposomes, and their spectra were analyzed in the range +73 to-14-degrees-C. For the 18:0 fatty acid derivative, first spectral moments, M1, were calculated and plotted as a function of temperature for each sample composition. Spectral inspection for regions of phase coexistence, in conjunction with consideration of M1 curves, permitted derivation of phase diagram boundaries which were then refined using spectral subtraction techniques. The phase diagram for galactosylceramide with short fatty acid in SOPC was compared to the corresponding phase diagram for its long-chain analogue, derived previously in the same fashion [Morrow, M. R., Singh, D., Lu, D., & Grant, C. W. M. (1992) Biochim. Biophys. Acta 1106, 85-93]. The binary phase diagrams referred to above, which reflect the behavior of short- and long-chain glycolipids in a common phospholipid host matrix, displayed important similarities and differences. In fluid membranes, the behavior appeared to be remarkably alike, as reflected in superimposable fluidus curves over the concentration range studied. Similarity was also seen in gel phase membranes at low glycolipid concentration (<20 mol %), although the long-chain species demonstrated greater capacity to stabilize the gel phase membrane against the gel/fluid transition. At higher mole fractions (>20 mol %), the long-chain glycolipid displayed a much greater tendency to gel/fluid phase separation and also to gel phase immiscibility which was not seen for the short-chain analogue. Both glycolipids were miscible in the liquid-crystal phase. A tendency for glycosphingolipid to order phosphatidylcholine membranes was evident in all samples. Partial synthesis of N-lignoceroyl[d7]-galactosylceramide, selectively deuterated on carbon atoms near the methyl terminus, permitted analysis of the novel H-2 NMR spectrum of its perdeuterated analogue and refinement of its phase diagram. The availability of an essentially complete spectral analysis also permitted derivation and comparison of order parameter profiles for short- and long-chain glycolipid fatty acids in the fluid 18-carbon fatty acid host matrix. The gradient of order parameter with respect to distance from the sugar headgroup for the 24-carbon glycolipid was comparable to that for the 18-carbon analogue to the depth of C-14, beyond which point its slope became sharply reduced.