Assay of proteasome proteolytic activity

Chymotrypsin-, trypsin- and caspase-like (CT-, T- and C-like, respectively) peptidase activities of the proteasomes were determined using, respectively, Suc-LLVY-AMC, Ac-RLR-AMC and Z-LLE-AMC substrates (all purchased from Enzo Life Sciences) at a concentration of 0.25 mM in 50 mM Tris-HCl, pH 7.5, containing 5 mM MgCl₂, 40 mM KCl, 1 mM DTT, 1 mM ATP at 37 °C for 45 min as described previously [66, 67]. The reaction was stopped by adding an equal volume of stop solution (0.1 M sodium chloroacetate, 30 mM sodium acetate, 25 mM acetic acid, pH 5.0). Proteasome activity was monitored by measuring free AMC fluorescence, following subtraction of background fluorescence, using a VersaFluor fluorometer (BioRad) with an excitation wavelength of 365 nm and an emission wavelength of 440 nm or a FLUOstar Omega fluorometer (BMG Labtech) with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The amount of liberated AMC was determined as fluorescence intensity. For a specificity control, the purified proteasomes were treated with 100 μM proteasome inhibitor MG132 or vehicle (DMSO).