Protease-nicked theta-toxin of Clostridium perfringens, a new membrane probe with no cytolytic effect, reveals two classes of cholesterol as toxin-binding sites on sheep erythrocytes.
A nicked theta-toxin (C theta), obtained by limited proteolysis with subtilisin Carlsberg, causes almost no hemolysis while it retains a nearly intact cholesterol binding site below 20 degrees C. Neither electron microscopic evidence for the formation of arc- and ring-shaped structures on the membrane nor toxin-stimulated influx of extracellular Ca2+ are detected in C theta-treated cells below 20 degrees C. Thus, event(s) in the lytic process are responsible for the temperature dependency of hemolysis, which is also supported by the observation that C theta requires higher Arrhenius activation energy for hemolysis than the native toxin. Using C theta as a probe due to its high affinity for membrane cholesterol without causing any obvious membrane changes, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes. Both binding sites disappear by simultaneous treatment of the cells with sublytic doses of digitonin. Furthermore, C theta 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 an 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.
D000818:Animals; D001427:Bacterial Toxins; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000097:blood; D002784:Cholesterol / Q000097:blood*; D003016:Clostridium perfringens; D004910:Erythrocyte Membrane / Q000032:analysis*; D006460:Hemolysin Proteins; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D008563:Membrane Lipids / Q000097:blood*; D008854:Microscopy, Electron; D010447:Peptide Hydrolases; D051381:Rats; D012756:Sheep; D013696:Temperature
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008563:Membrane Lipids; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol; D010447:Peptide Hydrolases; D002118:Calcium