Analysis of sites that are cleaved in various substrate polypeptides (Table 2) facilitated the development of rapid fluorogenic and chromogenic assays for caspase activity. Because the caspases have overlapping cleavage site specificities (Table 1), these assays are not specific for individual caspases. This potential problem is best illustrated by the data of Talanian et al. (32) who analysed the Km and kcal of purified caspases acting on a variety of low molecular weight substrates. This analysis revealed that caspases-3 and -7 preferred the p-nitroaniline (pNA)-coupled peptide Ac-DEVD-pNA (Km = 11 or 12 |aM, respectively); caspase-1 preferred Ac-YEVD-pNA (Km = 7.3 p,M); and caspase-6 preferred Ac-VEID-pNA (Km = 30 |xM). These preferences were not, however, absolute. Caspase-3, for example, displayed a kcJKm (a measure of catalytic efficiency) of 21.8 X 104 for Ac-DEVD-pNA, 6.1 X 104 for Ac-VEID-pNA, and 3.9 X 104 for Ac-YEVD-pNA, indicating that efficiencies at cleaving the various substrates differed by only a factor of 5. Aside from the inefficient cleavage of Ac-DEVD-pNA by caspase-6, the other caspases showed similar promiscuity. Accordingly, the cleavage of small peptide-derived substrates in cell extracts is difficult to attribute to individual caspases.
In addition to tetrapeptide or pentapeptide substrates, in vitro translated proteins have also been used to assay caspase activity in vitro (33-35). The work of Andrade et al. (5) has demonstrated the feasibility of measuring the Km and kcat using these more relevant macromolecular substrates. Recent data suggest that caspase cleavage preferences derived solely from the study of tetrapeptide-based substrates might not accurately predict cleavage sites in substrate polypeptides (36), raising the possibility that macromolecular substrates might still have an important role in the study of caspases. On the other hand, the disadvantages of radiolabeled macromolecular substrates include the need to perform both in vitro transcription/translation reactions to generate substrates and SDS-PAGE followed by fluorography to analyse results. In short, this labour-intensive approach is not suitable for high throughput screening. Accordingly, we provide a protocol for a tetrapeptide-based assay.
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