Effect ofClostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane

Summary Human diploid embryonic lung fibroblasts were cultivated in Eagle's Minimum Essential Medium and labeled with³H-uridine. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane. The assay method described is simple, sensitive and rapid and allows quantitative estimation of changes in membrane permeability before any morphological damage is observed microscopically. Crude commercial preparations of phospholipase C (E.C. 3.1.4.3.) (40 g/ml) were highly active on the cell membrane but most of the membrane damaging activity was found to be due to contaminating theta-toxin. However, also highly purified phospholipase C caused a membrane damage as measured by release of isotope through the plasma membrane. The release could be increased by including an optimal concentration of calcium ions in the incubation buffer, by treating the cells in a hypotonic medium and by simultaneous treatment with sublytic concentrations of Triton X-100. To our knowledge this is the first report of membrane damage on a live, intact, metabolizing human diploid cell caused by a highly purified phospholipase C. The results are in agreement with a dynamic membrane structure with the polar groups of a part of the phospholipids accessible at the membrane surface.