Fig. 1.
Cholesterol stimulates the pore-forming activity of ClyA. (A) Turbidity assay to determine activity of ClyA is performed by measuring the optical density of rabbit erythrocytes (black) with time after addition of ClyA. Proteolytic shaving of erythrocyte membranes (shaved RBC; red) did not change the t1/2 of the lysis (Inset) or the extent of lysis. Solid lines represent Boltzmann sigmoid fits to the data. (B) Partial removal of cholesterol from erythrocytes by treatment with MβCD (red) increases the t1/2 for lysis in turbidity assays by more than 100-fold compared with untreated erythrocytes (black). (C) Vesicle dye leakage kinetics of ClyA for DOPC (green) and DOPC with 30% cholesterol (blue) and 50% cholesterol (gray) concentrations are shown. Solid lines represent single exponential fits to the leakage data. The dye leakage t1/2 in presence of cholesterol is reduced by approximately threefold for 30–50 mol % cholesterol (Inset). Error bars represent SD of at least three experiments.