On searching the databases for genes related to that for caspase-7, a cDNA clone was identified that encodes a novel 416-amino-acid protein with a predicted molecular mass of 46 kDa. The major difference beween caspase-9 and other family members is the active-site pentapeptide QACGG, in which a Gly is found instead of the usual Arg. Pro-caspase-9 contains a long N-terminal putative prodomain with high similarity to the prodomains of CED-3 and caspase-2 (Srinivasula et al., 1996; Duan et al., 1996).

Northern-blot analysis revealed the presence of multiple mRNA species, suggestive of alternative splicing. High levels of expression of caspase-9 are found in the heart, testis and ovary. Overexpression of caspase-9, but not of a mutant in which the catalytic Cys was replaced with an Ala, induced apoptosis in MCF7 cells. Pro-caspase-9 contains to potential processing sites between its large and small subunits, P³¹²EPD¯ A³¹⁶ and D³²⁷QLD¯ A³³¹. The latter motif is similar to the DEVD¯ G site in PARP, suggesting that caspase-9 may be activated by caspase-3, while the former motif may be a potential granzyme B cleavage site, as it contains an acidic residue in the P₃ position.

Using in vitro mutagenesis, it was demonstrated that both caspase-3, generating two products of molecular masses 37 kDa (p37) and 10 kDa (p10) and Granzyme B cleaved pro-caspase-9 at both sites. There is a marked preference for Asp-315 over Asp-330, generating an active enzyme capable of cleaving PARP to its signature fragment of 85 kDa. In some, but not all, studies the prodomain of caspase-9 was removed (Srinivasula et al., 1996; Duan et al., 1996). The ability of caspase-3 to activate pro-caspase-9 suggests that the latter is downstream of caspase-3 and, as such, may be responsible for some of the later changes seen in cells undergoing apoptosis.