Sly amc

Calpain proteolytic activity was measured by hydrolysis of the synthetic calpain substrates, sLY-AMC (N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin; Cayman) and Ac-LLY-AFC (Ac-Leu-Leu-Tyr-7-amino-4-trifluoromethylcoumarin; Fisher) (Wert et al., 2015 (link)). Briefly, 10 μM or 0.25 μM of purified calpain was added to a reaction buffer containing 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 2 mM DTT, and 0.1 to 100 mM CaCl₂ or 5 mM EDTA and incubated on ice prior to reaction in 96-well plates (100 μL per reaction). Varying concentrations of substrate (10 μM to 1 mM) were added and the reaction was incubated at 37°C for 1 hour on a fluorimetric plate reader (Tecan Spark, Männedorf Switzerland). All experiments were performed in triplicate. The amount of AMC or AFC released during the experiment was calculated based on a standard curve of AMC or AFC concentrations (from 2 nM to 5 μM) and kinetic parameters were calculated by direct fitting to the Michaelis-Menten equation in GraphPad Prism 8. Inhibition experiments were carried out in similar manner with either E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (Sigma) or the human CAST B27 domain (residues 184–210; Calbiochem) in the presence of 0.2 mM Ac-LLY-AFC. Reaction rates at various inhibitor concentrations (10 nM to 10 μM) were normalized and fit to the Hill equation in GraphPad Prism 8.