Glucose permeability of liposome vesicles prepared with sterol extracts from fenpropimorph-grown fungi

pressure are used to vary fluidity, and from Lianos et al. (1980) it may be postulated to report the outer, polar regions of bilayers (Macdonald et ul., 1987). The incorporation of oleic acid into microsomal membranes may be expected to fluidize the bilayer interior and only secondarily affect the region in which pyrene is thought to preferentially partition. Hence, the E / M ratio obtained from such membranes should be increased. The polarization of DPH, which emits signals from the bilayer interior, should be markedly reduced. The incorporation of stearic acid may be expected to exert little or no effect on the E / M ratio, but it should increase DPH polarization. To test these predictions rat liver microsomes were prepared as previously described (Macdonald et al., 1987) in a solution containing (mM): sucrose, 220; Tris/HCI, 22.8; EDTA, 1.07; dithiothreitol, 1.0; pH 7.4. To a stirred suspension of A,,,,=O.l, oleic acid or stearic acid was added in ethanol, at room temperature, giving a final concentration of 0.1 mM in the suspension. The concentration of ethanol was 50 mM in both treated suspensions and in controls, the latter receiving an appropriate addition of ethanol only. After 60 min equilibration pyrene was added, in the ratio of 25 mMpyrene per mol phospholipid phosphate. DPH was added to other suspensions and after a further 30 min equilibration at room temperature, aliquots were degassed in a vacuum and read at 25°C on a Perkin Elmer L-S-3 spectrometer, as in Macdonald et al. (1987). The following corrections were applied. Endogenous fluorescence and scattering effects and, separately, the fluorescence emitted by the aqueous supernatants, were appropriately deducted from the gross data obtained from each of the fluophors. Membranes exposed to oleic acid incurred a large combined correction ( > 20%) in the case of pyrene. A third correction was also required for pyrene the microsome-supernatant partition coefficient of which was altered by the presence of exogenous fatty acids. By extracting the pyrene in microsomal pellets, the reduced concentration of pyrene in the microsomes treated with fatty acids was determined. From a plot of the E / M ratio against the concentration of pyrene in the microsomes, the slope of which was unaffected by oleic acid, a correction was applied to the gross E / M ratio. In the case of oleic acid this was 12% and for stearic acid it was zero. Using 14C-labelled fatty acids as markers, approximately 90% of the added stearic and oleic acids partitioned into the microsomes and 83% of this remained as free fatty acid after the 1 h incubation. Incorporation of label into phospholipids was < 1% and into triacylglycerol was 13% and 15%, respectively, for stearic and oleic acids. Table 1. The I’yrene E / M ratio and the sieady-siate ,fluorescence polarization of DI’H in rat liver microsomes treaied wiih oleic mid stearic acid