In order to monitor the membrane fluidity of cells without perturbation by an introduced probe, we developed a method for large-scale preparation of 2H-labeled
melanoma cells for a 2H NMR study by incubating
melanoma cells with [18,18,18-2H3]
stearic acid/
phosphatidylcholine liposomes for 2 h at 37 degrees C. It turned out that this treatment did not significantly change the cell viability, lipid metabolism or membrane fluidity. The 2H from C-18 of
stearic acid is dominantly located at the original position of the
fatty acid in the 2H-labeled membrane vesicles, as studied by a tracer experiment with [1-14C]
stearic acid. We found that three to four 2H-labeled species were present at 19 degrees C in 2H NMR spectra of the 2H-labeled membrane vesicles prepared from
B16 melanoma cells. The extent of peak-splittings due to 2H-quadrupole interaction decreased as the temperature rose, and a definite point of phase transition was not observed. At elevated temperature, 2H-labeled
lipids undergo fast exchange between the bilayer and an isotropic phase such as oil phase of
triolein or inverted
micelles in
lipid polymorphs. We further analyzed the change of membrane organization in mouse
B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), which strongly inhibited melanogenesis. The magnitude of the quadrupole splitting at 19 degrees C in membranes from TPA-treated cells was significantly less (40%) than in the untreated control. This is mainly explained by decreased molecular ordering (fluidity) due to the increased amount of
unsaturated fatty acids in the membranes of TPA-treated cells.