Equilibrium Uptake of D-Glucose by Osmotically Stressed Unilamellar Phospholipid Vesicles

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The assumptions inherent in the use of osmotic manipulation to determine the extent of solute binding to brush border membrane vesicles (the ideal osmotic responsiveness of the vesicles and the independence of solute binding from the incubation medium osmotic pressure) were examined in a model system (large unilamellar lipid vesicles). The equilibrium uptake of D-glucose by unilamellar vesicles composed of egg lecithin (PC), phosphatidic acid (PA), and cholesterol (Chol) was measured as a function of the osmotic concentration of the incubation medium. The variation of the encapsulated aqueous volume of PC:PA and PC:PA:Chol vesicles with the osmotic stress was directly determined by a fluorescence self-quenching technique. Encapsulated volume changes of both PC:PA and PC:PA:Chol vesicles were found to be resistant to the osmotic stress, exhibiting positive deviations from ideal behavior. Equilibrium uptake experiments with these vesicles showed that glucose was taken up in excess of that amount predicted on the basis of the encapsulated volume when the vesicles were subjected to osmotic stress <0.25 osmol/kg. At osmotic stresses >0.75 osmol/kg, equilibrium uptake could be predicted solely on the basis of the encapsulated volume. These results, based on a model vesicle system, strongly suggest that osmotic manipulation may be an inappropriate method to assess the extent of solute binding to natural membrane vesicle preparations, such as brush border membrane vesicles, without more direct evidence.