Purified calpain removes the prodomain of caspase-9 and caspase-3 via specific limited proteolysis. Calpain (100 nmol/L) was reacted with 35S-procaspase-9 (5 μL; A) or recombinant procaspase-3 (10 μmol/L; B) in the absence and presence of calpeptin (1 μmol/L) or ZVAD-fmk (1 μmol/L) and the products were analyzed by SDS-PAGE and autoradiography (A) or staining with Coomassie Blue (B). To determine whether calpain activates procaspase-3, we treated the procaspase with calpain and then measured caspase-3 activity by monitoring DEVD-AFC hydrolysis as shown in (C). The graph shows DEVD-AFC hydrolysis by procaspase-3 (1), procaspase-3 reacted with calpain (2), granzyme B (3), or calpain then granzyme B (4) (error bars represent the SEM, N = 3). Columns 5 and 6 show the DEVDase activity of calpain and granzyme B, respectively. In (D), purified procaspase-9 and procaspase-3 were digested with calpain and the products sequenced by Edman degradation. The insets show the calpain cleavage sites. C287 and C163 denote the caspase-9 and caspase-3 active site cysteines. The shaded areas between the caspase large and small subunits represent the inter-domain linker that is removed upon caspase activation.
