Figure 7 | Cell Death & Differentiation

Figure 7

From: Isolation of AmphiCASP-3/7, an ancestral caspase from amphioxus (Branchiostoma floridae). Evolutionary considerations for vertebrate caspases

Figure 7

AmphiCASP-3/7 has a substrate specificity similar to caspase-7. (A) Fifty micrograms of bacterial lysates from clones expressing recombinant human caspase-3 (hCASP3), human caspase-7 (hCASP7), amphioxus AmphiCASP-3/7 (CASP37) and mutated AmphiCASP-3/7 (C205S) were assessed for the ability of cleave different fluorogenic substrates for 7 h at 37°C. AmphiCASP-3/7 extracts were also either left untreated or pre-incubated with 20 μM of the inhibitors DEVD-fmk and VAD-fmk. As a control, extracts from cultures transformed with the empty vector were included (Pet21a). Caspase activity was monitored measuring the -afc release on Ac-DEVD-afc (black bars) Ac-VEID-afc (white bars) and Ac-IETD-afc (grey bars) substrates as arbitrary fluorescence units in a Bio-Tek FL 600 fluorimeter (Izasa, Spain) at 360 nm (40 nm bandwidth) of excitation and 530 nm (25 nm bandwidth) of emission. Data (±S.E.M.) were derived from one experiment performed in triplicate. (B) Different concentrations (μg) of bacterial lysates expressing recombinant human caspase-3 (filled squares), caspase-7 (filled circles) and AmphiCASP-3/7 (open circles) were assessed for their ability to process Ac-DEVD-afc (left plot) and Ac-VEID-afc (right plot) as described in (A)

Back to article page