Acetyl ile glu pro asp amc

The enzyme activities were determined using acetyl-Ile-Glu-Pro-Asp-AMC (50 µM) and Z-Gly-Pro-Arg-AMC (200 µM; both from Bachem) for granzyme B and A, respectively. The following assay buffers were used: 50 mM Tris-HCl and 100 mM NaCl, pH 7.4 (for granzyme B) and 20 mM Tris and 150 mM NaCl, pH 8.1 (for granzyme A). Whole-cell lysates were first activated in assay buffer for 30 min at 37 °C. The substrate was then added, and the formation of fluorescent products was measured continuously on a microplate reader Infinite M1000 (Tecan, Männedorf, Switzerland).

Enzyme activities were determined using the following substrates: 70 µM H-Gly-Phe-7-amino-4-methylcoumarin (AMC) (Bachem) for cathepsin C, 20 µM H-Arg-AMC (Bachem) for cathepsin H, 50 µM Z-Phe-Arg-AMC for cathepsin L (Bachem), 50 µM acetyl-Ile-Glu-Pro-Asp-AMC for granzyme B (Bachem), and 200 µM Z-Gly-Pro-Arg-AMC for granzyme A (Bachem). The following assay buffers were used: 25 mM MES, 100 mM NaCl, 5 mM cysteine, pH 6 (for cathepsin C); 100 mM MES, 2 mM EDTA, 5 mM cysteine, pH 6.5 (for cathepsins H and L); 50 mM Tris-HCl, 100 mM NaCl, pH 7.4 (for granzyme B); and 20 mM Tris, 150 mM NaCl, pH 8.1 (for granzyme A). Whole-cell lysates were first activated in assay buffer for 15 min at RT for cathepsins or for 30 min at 37 °C for granzymes. The substrate was then added, and formation of fluorescent products was measured continuously on a microplate reader Infinite M1000 (Tecan, Männedorf, Switzerland). To determine cathepsin L activity, 5 µM of irreversible inhibitor of cathepsin B, CA-074 (Bachem), was added before the addition of substrate.