Active Cystatin A (CSTA)

Cystatin A (CSTA) is a member of family 1 of the cystatin superfamily. Like Cystatin B, it is an intracellular inhibitor regulating the activities of cysteine proteases of the papain family such as cathepsins B, H and L. For example, immunohistochemical analysis of Cystatin A and cathepsin L is a useful indicator for malignancy in human epidermal keratinocytes. The ratio of cathepsin B and Cystatin A can be used in the differential diagnosis and treatment of patients with prostate carcinoma. The activity of recombinant pig Cystatin A was measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC in the assay buffer 50 mM Tris, pH 7.0. Papain was diluted to 500 ug/ml in activation buffer 50 mM Tris, 5 mM DTT, pH 7.0 and incubated at room temperature for 15 minutes. The activated papain was diluted to 100 ug/ml in the assay buffer and 20 ul different concentrations of recombinant pig Cystatin A (MW: 40.9 KD) was incubated with 20 ul 100 ug/ml papain at 37 ℃ for 10 minutes. Loading 50 µL of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50 µL of 200 µM substrate. Include a substrate blank containing 50 µL of assay buffer and 50 µL of 200 µM substrate. Then read at excitiation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant pig Cystatin A significantly decreased papain activity. The inhibition IC50 was <300 nM.