Figure 4
Phospholipid requirements of PhlA hemolytic and cytotoxic activities. (A) Human RBC were mixed with various concentrations of His-PhlA in the absence (open circles) or presence of lecithin (filled circles) or phosphatidylcholine (filled squares) and incubated at 37°C for 1 h. (B) Human RBC were mixed with various concentrations of lysophosphatidylcholine (LPC) and incubated at 37°C for 1 h. (C) Products of the reaction of PC with (+) or without (-) His-PhlA were analyzed by thin-layer chromatography. (D) Human (circles), sheep (triangles), and horse (squares) RBC were mixed with 8.3 μM PhlA (filled symbols) or no PhlA (open symbols) and incubated at 37°C for 1 h in the presence of various concentrations of lecithin with 2 mM CaCl2. (E) HeLa and 5637 cells were exposed to various concentrations of His-PhlA for 1 h in the presence of lecithin. His-PhlA cytotoxicity was evaluated with a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Open and filled circles show HeLa and 5637 cells, respectively. Values are averages ± SE from three independent experiments. (A), (B), and (D) Results are expressed as percent lysis compared with lysis of RBC in distilled water, as in the contact hemolysis assay (Fig. 1).