Mr. Morrow et al., GLYCOSPHINGOLIPID HEADGROUP ORIENTATION IN FLUID PHOSPHOLIPID CHOLESTEROL MEMBRANES - SIMILARITY FOR A RANGE OF GLYCOLIPID FATTY-ACIDS, Biophysical journal, 69(3), 1995, pp. 955-964
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.