FIG. 5.

The cytotoxic activity in EP contains lipase and protease sensitive components. (A) A 50-μg/ml S. flexneri EP solution was incubated for 4 h at 37°C either alone (closed circles), with uncoupled agarose beads (open circles), or with 22 U of wheat germ type 1A triacylglycerol lipase-agarose beads per ml (closed squares). Beads were removed by centrifugation, and the supernatants were tested for cytotoxicity on THP-1 cells at the indicated dilutions. Percent cytotoxicity in panels A and B was determined by an LDH release assay. (B) A 200-μg/ml sBLP solution was treated and assayed as described for panel A. (C) SDS-PAGE analysis of the cytotoxic activity in S. flexneri EP and PK-treated S. flexneri EP. One hundred sixty-six micrograms of S. flexneri EP (black bars) or 166 μg of PK-treated S. flexneri EP (gray bars) was resolved by Tris-Tricine SDS–16.5% PAGE. Eleven 5-mm gel sections (fractions) were made per lane, extracted with octyl glucoside, and tested for cytotoxic activity by an LDH release assay on cycloheximide-treated THP-1 cells by serial dilution directly into tissue culture media. Activity is defined as 1/(dilution yielding 50% maximal cytotoxic activity). Peptide molecular mass markers (in kilodaltons) are shown on the left.