Protease‐nicked O‐toxin of Clostridium perfringens, a new memnrane probe with no cytoltic effect, revcals two classes of cholesterol as toxin‐binding sites on sheep erythrocytes

Abstract

A nicked O-toxin (Co), Obtaned by limited proteolysis with subtilisin Carlsberg, causes almost no hemolysis while it retains a nearly intact cholesterol binding site below 20°C. Neither electron micreoscopic evidence for the formation of arc- and ring-shaped structures on the membrane non toxin-stimulated influx of extracellular Ca²⁺ are detected in Co-treated cells below 20°. Thus, event(s) in the lytic process are responsible for the temperature deoebdency of hemolysis, which is also supported by the observation that Co requires higher Arrhenius activation energy for hemolysis than the native toxin. Using Co as a probe due to its high affinity ofr membrane cholesterol without causing any obvious membrane change, we demonstrated the possible existence of high-and low-affinity sites for o-toxin on sheep erythrocytes. both binding sites desappear by simulataneous treatment of the cell with sublytic doses of digitonin. Furthermore, Co Binds only to cholesterol among the chloroform/methanol- extractable, lipid components of sheep and human erythrocytes but not to the protein components derived from them. these results strongly suggest that cholesterol is and essential component of the both high- and low-affinity sites, and also imply that the modes of existence of cholesterol in the red cell membrane are heterogeneous.