GLYCOSPHINGOLIPID HEADGROUP ORIENTATION IN FLUID PHOSPHOLIPID CHOLESTEROL MEMBRANES - SIMILARITY FOR A RANGE OF GLYCOLIPID FATTY-ACIDS

Galactosylceramide (GalCer) was labeled for nuclear magnetic resonance (NMR) spectroscopy by replacement of a hydrogen atom at C-6 of the ga lactose residue with deuterium. Wideline H-2 NMR of [d(1)]GalCer permi tted consideration of a mechanism traditionally entertained for cell s urface recognition site modulation: that the nature of the fatty acid attached to the sphingosine backbone of glycosphingolipids (GSLs) impo rtantly influences carbohydrate headgroup orientation. Comparison was made among various glycolipid fatty acids by altering hydroxylation, s aturation, and chain length. Studies were carried out in unsonicated b ilayer membranes mimicking several important characteristics of cell p lasma membranes: fluidity, low GSL content, predominant [sn-2]monounsa turated phosphatidylcholine (PC) (1-palmitoyl-2-oleoyl PC), and the pr esence of cholesterol. Spectroscopy was performed on samples over a ra nge of temperatures, which included the physiological. H-2 NMR spectra of [d(1)]GalCer having 18-carbon saturated fatty acid (stearic acid), cis-9-unsaturated fatty acid (oleic acid), D- and L-stereoisomers of alpha-OH stearic acid, or 24-carbon saturated fatty acid (lignoceric a cid), were importantly similar. This argues that for GSLs dispersed as minor components in fluid membranes, variation of the glycolipid fatt y acid does not provide as much potential for direct conformational mo dulation of the carbohydrate portion as has sometimes been assumed. Ho wever, there was some evidence of motional differences among the speci es studied. The H-2 NMR spectra that were obtained proved to be more c omplex than was anticipated. Their features could be approximated by a ssuming a combination of axially symmetric and axially asymmetric glyc olipid motions. Presuming the appropriateness of such an analysis, at a magnetic field of 3.54 T (23.215 MHz), the experimental spectra sugg ested predominantly asymmetric motional contributions. At the higher f ield of 11.7 T (76.7 MHz, equivalent to a proton frequency of 500 MHz) , spectra indicated dominance by axially symmetric rotational modes. T here was also evidence of some bilayer orientation in the stronger mag netic field. The unusual observation of spectral differences between t he two magnetic field strengths may involve a diamagnetic response to high field on the part of some liposome physical characteristics.