Macroscopic Consequences of the Action of Phospholipase Con Giant Unilamellar Liposomes

AbstractMacroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10–100μm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X=0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, Tm, i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below Tm. These results are supported by the enhanced activity of PC-PLC upon exceeding Tm of DMPC large unilamellar vesicles (diameter ∼0.1μm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below ∼30mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value