Spectroscopical characteristics of galactosylceramide-pyrene and ceramide-pyrene incorporated in model and in clathrin coated vesicles.

In order to control the ability of two pyrene-sphingolipids (ceramide-pyrene (Cpyr) and galactosylceramide-pyrene (GCpyr)) to monitor the changes in the lipid bilayer dynamics of cellular membranes, their incorporation in three populations of clathrin coated vesicles which differ in their structural characteristics (Bomsel et al. (1988) Biochemistry 27, 6808-6812) was studied by both absorbance and fluorescence spectroscopy. The method of injection of an ethanolic solution of probe was used. The analysis of the spectra recorded after injection into a free-membrane buffer allowed to discriminate two dispersion states (micellar or aggregated) of the probes. The micellar state was identified as the one suitable for the incorporation within the bilayer. Rising the temperature up to 18 degrees C for a membrane labeling with GCpyr and to 37 degrees C for a membrane labeling with Cpyr was found to be necessary because it allowed to slow down the aggregation process which inhibited the incorporation within the lipid bilayer. The excimer/monomer (E/M) fluorescence intensities ratio of GCpyr was found to be characteristic of each population of coated vesicles. Cpyr could not be used as a diffusion probe because it partly aggregated during the cooling step necessary to establish the E/M versus temperature plot in the heating mode. An important point which arises from these data is that the use of absorbance spectroscopy can avoid misinterpretation of the pyrene derivatives fluorescence spectra in terms of diffusion.