Interaction ofPseudomonas aeruginosa cytotoxin with plasma membranes from Ehrlich ascites tumor cells

Summary

Biologically active¹²⁵I-cytotoxin fromPseudomonas aeruginosa binds to plasma membranes from Ehrlich ascites tumor cells in a saturable manner. The Scatchard plot indicated a single binding site with a capacity of 260 pmoles/mg of membrane protein and aK _D of 2×10⁻⁸ M. Specific binding was dependent on temperature, pH and ionic strength. Thus constant levels of bound¹²⁵I-cytotoxin were attained either within 30 min at 30°C or within 3 h at 4°C. Binding was 30-fold higher at 4°C vs 30°C and 2-6-fold higher at pH 5.3 vs pH 8.3. Binding was not effected by 50 mM sugar or sialic acid. 300 mM sucrose, however, instead of phosphate buffer, reduced binding by 50%. Pretreatment of plasma membranes with trypsin or papain led to a significant decrease in¹²⁵I-cytotoxin binding. A pretreatment with phospholipase C or D had no effect, whereas phospholipase A₂ induced a decrease by 34%. The collected data suggest that the binding site for¹²⁵I-cytotoxin within the plasma membrane from Ehrlich ascites tumor cells is a membrane protein.

Correlation of¹²⁵I-cytotoxin binding and membrane action of the unlabelled cytotoxin can be observed through (a) increased lowering of the cellular K⁺ and Na⁺ gradient by decrease of medium pH, (b) decreased toxicity after substitution of ions by sugar, and (c) increased breakdown of cellular cationic gradient after temperature shift from 4°C to 37°C.