Cleavage (A) and activation (B) of procaspase-3 by P. gingivalis-excreted gingipains. (A) Incubation of pure procaspase-3 with caspase-8 (4 or 8 ng) or culture supernatants ('sup') of wild type (WT) or triple gingipain mutant (MT) P. gingivalis (20 or 40 ng protein content, labeled over lanes) were done as described under Experimental Procedures. The products were analyzed by SDS-PAGE and silver-staining. The full-length procaspase-3 (34 kDa) and the processed active fragments (17 and 12 kDa) are marked by open and closed arrowheads, respectively. (B) Activity of the processed caspase-3 generated in Panel A was measured using a colorimetric peptide substrate described under Experimental Procedures. The enzymes used to cleave procsapase-3 are: 20 ng of wild type P. gingivalis sup (Triangle); 4 ng caspase-8 (Squares); 20 ng of gingipain mutant P. gingivalis sup (Open circles); 20 ng of wild type P. gingivalis sup, preheated at 65°C for 15 min (Diamonds); none (Closed circles). The error bars are derived from three independent experiments.