Parameters affecting fusion between liposomes and synaptosomes. Role of proteins, lipid peroxidation, pH and temperature

Abstract

We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mm)/ Fe²⁺ (2.5 μm)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25°C. This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggregation, as well as for the role of electrostatic forces in this process, at mild acidic pH.