Determination of Toxin-induced Leakage of Different-size Nucleotides Through the Plasma Membrane of Human Diploid Fibroblasts

Human diploid lung fibroblasts were treated with cytolytic bacterial toxins and the nature of the membrane damage was investigated. [3H] uridine was used for differential labeling of cytoplasmic components of small or large molecular size. Two principal size categories were achieved by labeling the fibroblasts in either early growth phase or stationary phase, a high-molecular weight ribonucleic acid label and a low-molecular-weight nucleotide label. The size of the labeled molecules was determined by perchloric acid precipitation and gel chromatography. Leakage of labeled molecules of different size indicated the size of the "functional pores" in the plasma membrane caused by the test substance. The nonionic detergent Triton X-100 produced large functional pores in the fibroblast membrane as evidenced by rapid leakage of both large and small labeled molecules. Theta-toxin from Clostridium perfringens and the polyene antibiotic filipin both gave rise to considerably small functional pores in the plasma membrane. Although small molecules easily passed the treated membrane, large molecules could not escape from the cells even after prolonged treatment with these substances or by increasing their concentration. By the contrast, the leakage profiles obtained with melittin from bee venom or with delta-toxin from Staphylococcus aureus in each case suggested the formation initially of pores of intermediate size that increased upon prolonged incubation or when higher concentrations were used.