When erythrocyte membranes were treated with
cereolysin, negatively stained and examined by electron microscopy, ring and
arc-shaped structures were observed in the membrane. The outside diameter of the rings varied from 33 to 50 nm with a border thickness of 6.7 to 8.3 nm. The
arcs varied in length from 33 to 170 nm with a border thickness of also 6.7 to 8.3 min. When right-side-out erythrocyte ghosts which had been treated with
cereolysin were examined by electron microscopy after freeze-fracture, structures with a diameter of 31 to 63 nm were seen in the fracture face of the exoplasmic half of the membrane, but no alterations were visible in the fracture face of the protoplasmic half of the membrane bilayer. Thus the ring structures did not appear to form holes through the membrane. At
cereolysin concentrations above 6 microgram/ml rings and
arcs were seen when purified toxin alone was examined. At or below 6 microgram/ml toxin rings and
arcs were seen only if toxin was incubated with free or membrane-bound
cholesterol. Our interpretation is that
cereolysin tends to aggregate into ring and
arc-shaped structures, and that the tendency to aggregate is increased by
cholesterol. Rings and
arcs were not seen when erythrocyte ghosts were treated with low, but lytic amounts of
cereolysin that significantly altered the premeability of the ghosts.