Study on the interaction of theta-toxin with membrane cholesterol

1.A hemolysin，theta-toxin，produced by Clostridium perfringens has one thiol group which is essential for its hemolytic activity。Modification of the residue with thiol-blocking agents reduced the binding affinity of the toxin to sheep erythrocytes to1/100that of intact toxin，resulting in a failure of binding.Thus the failure of hemolysis is primarily ascribed to a decreased affinity of the socin for erythrocytes.2。The primary receptor for theta-toxin is the membrane cholesterol on the cell surface.The effect of theta-toxin on membrane fluidity was tested in hepatocyte preparations.Incubation of hepatocytes for 5min with 600 or 2000 units/ml of theta-toxin resulted in a significant increase(32and46%，respectively)of D_-as compared to the controls of the same age group when measured by fluorescence recovery after photobleaching technique.Various tests like heat denaturation or cholesterol saturation of theta-toxin before its application revealed that the increase of D__-is a specific toxin effect due most probably to the reaction of theta-toxin with cholesterol.3。A nicked theta-toxin(Ctheta)，obtained by limited proteolysis with subtilisin carlsberg，causes almost no hemolysis while it retains nearly intact cholesterol binding site below 20°C.Using Ctheta 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 sheep。Ctheta 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.

1. A hemolysin, theta-toxin, produced by Clostridium perfringens has one thiol group which is essential for its hemolytic activity. Modification of the residue with thiol-blocking agents reduced the binding affinity of the toxin to sheep erythrocytes to 1/100 that of intact toxin, resulting in a failure of binding. Thus the failure of hemolysis is primarily ascribed to a decreased affinity of the socin for erythrocytes.2. The primary receptor for theta-toxin is the membrane cholesterol on the cell surface. The effect of theta-toxin on membrane fluidity was tested in hepatocyte preparations. Incubation of hepatocytes for 5 min with 600 or 2000 units/ml of theta-toxin resulted in a significant increase (32 and 46%, respectively) of D__- as compared to the controls of the same age group when measured by fluorescence recovery after photobleaching technique. Various tests like heat denaturation or cholesterol saturation of theta-toxin before its application revealed that the increase of D__- is a specific toxin effect due most probably to the reaction of theta-toxin with cholesterol.3. A nicked theta-toxin (Ctheta), obtained by limited proteolysis with subtilisin carlsberg, causes almost no hemolysis while it retains nearly intact cholesterol binding site below 20゜C. Using Ctheta 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. Ctheta 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.