Apoptosis can be mediated by mechanisms other than the traditional caspase-mediated cleavage cascade. There is growing recognition that alternative proteolytic enzymes such as the lysosomal cathepsin proteases may initiate or propagate proapoptotic signals. Cathepsins are lysosomal enzymes that are also used as sensitive markers in various toxicological investigations. The Cathepsin D Activity Assay kit is a fluorescence-based assay that utilizes the preferred cathepsin D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labeled with MCA. Cathepsin D will cleave the synthetic substrate to release the quenched fluorescent group MCA, which can then easily be measured using a fluorometer or fluorescence plate reader at Ex/Em = 328/460 nm. The relative efficacy of test inhibitors are compared to the positive control inhibitor, Pepstatin A (IC50 < 0.1 nM). The Cathepsin D assay is simple, straightforward, and can be adapted to 96-well plate assays and is suitable for high throughput screening (HTS).

Figure 1: Typical Pepstatin A Inhibition Profile of Cathepsin D Activity. Blue line denotes an IC 50 value of 0.067 nM. Results were analyzed by a fluorescence plate.