GLYCOSPHINGOLIPIDS - H-2 NMR-STUDY OF THE INFLUENCE OF CERAMIDE FATTY-ACID CHARACTERISTICS ON THE CARBOHYDRATE HEADGROUP IN PHOSPHOLIPID-BILAYERS

Galactosylceramides bearing a variety of different pure fatty acid chains were H-2 labeled in the carbohydrate headgroup at C6 of the terminal galactose residue, for study by H-2 NMR. Fatty acids investigated included the 24-carbon saturated lignoceric acid, 18-carbon saturated stearic acid, cis-9,10-unsaturated oleic acid, and D- and L-stereoisomers of alpha-hydroxystearic acid. Headgroup-deuterated glycolipids were incorporated at 10 mol % into unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and H-2 NMR spectra were recorded at 65 and 40-degrees-C. Under these experimental conditions, the membranes studied were primarily in the liquid-crystalline phase. At a given temperature, spectra for deuterated galactosylceramides dispersed in the fluid phase were remarkably similar, regardless of the nature of the fatty acid attached to the glycolipid sphingosine backbone. In each case, the spectrum consisted of a superposition of two quadrupolar powder patterns of approximately equal intensity. The spectra may be interpreted as arising from equal populations of two stereoisomers (pro-R and pro-S) of the deuterated galactose hydroxymethyl function, which is undergoing rapid (> 10(6) s-1) interconversion among the possible rotamers about the C5-C6 bond of the sugar ring. Within experimental error, the only fatty-acid-induced spectral difference detected among these glycosphingolipids deuterated in the carbohydrate headgroup was in the species with alpha-hydroxy-substituted fatty acids. At 65-degrees-C, N-(D-alpha-hydroxy)stearoyl- and N-(L-alpha-hydroxy) stearoylgalactosylceramide gave rise to the same quadrupole splittings, but these differed marginally from the splittings observed for the other glycolipids studied. At 40-degrees-C, the DELTA-nu(Q) values, for the L-alpha-hydroxy species only, differed measurably from those of the other fatty acid derivatives. Our results would argue that, at least in the case of galactosylceramide in fluid phospholipid bilayers, very little change in the average glycosphingolipid carbohydrate headgroup orientation (probably less than a few degrees), and in the amplitude of orientational fluctuations about the bilayer normal, is induced by major alteration in the nature of the fatty acid attached to the sphingosine backbone. The effect of temperature on the carbohydrate portion, in the range studied, appeared to be confined largely to changes in motional order. At 65-degrees-C, freeze-fracture electron microscopy of the liposomal preparations examined in this work demonstrated totally fluid bilayer membranes in all cases. At 40-degrees-C, the phospholipid host matrices containing N-stearoylgalactosylceramide and those containing N-lignoceroylgalactosylceramide showed minor areas of gel-phase lipid, which seem likely to represent glycolipid-rich domains. This is a phenomenon not previously recorded by electron microscopy and was not seen for the other fatty acid derivatives at the same temperature.