H-2-NMR STUDY OF 2 PROBE-LABELED GLYCOSPHINGOLIPID-DERIVED SIGNALING MODULATORS IN BILAYER-MEMBRANES

We describe here the first report of sphingoid bases bearing non-pertu rbing H-2 probe nuclei. These were produced, by two different routes o f partial synthesis, to permit direct assessment of their arrangement and behaviour as minor components in membrane systems. Wideline H-2-NM R spectra of N, N-dimethylsphingosine with deuterated amino-methyl gro ups ([(2) H-6]dimethylsphingosine), and of lyso-dihydrogalactosylceram ide (lyso-GalCer) with deuterium nuclei at C-4,C-5 of the sphingosine backbone and at C-3,C-4 of the galactose ring ([H-2(4)]lyso-GalCer), w ere recorded in unsonicated, cholesterol-containing fluid bilayer memb ranes. The sphingolipid metabolites behaved as single populations of l ipid amphiphiles dispersed uniformly in the membrane and undergoing ra pid symmetric motion about their long molecular axes. This was the cas e throughout the pH ranges examined, which included values generally c onsidered for the cell cytoplasm. Spectra of [H-2(6)]dimethyl sphingos ine indicated that the methyl groups are equivalent on the NMR timesca le, and that the molecule's orientation and behaviour are largely unaf fected by pH over the range, 6 to 10.5. There was no spectral evidence of deprotonation of the tertiary amine function in this range. Simila rly, variation of pH between 6.4 and 8.9 had virtually no effect on th e average conformation and orientational order of lyso-GalCer at the l evel of C-4,C-5 in the sphingosine backbone. pH did, however, exert si gnificant control over the orientation of the galactose residue - the effect being most marked in the region of the sphingoid base pK(a). Th e lyso-glycolipid showed some evidence of being less motionally ordere d than the corresponding parent species, presumably as a result of rem oval of constraints imposed by the fatty acid.