3.3. Assay of Proteasome Proteolytic Activity

Chymotrypsin-like (CT-like) peptidase activity of the proteasomes was determined using Suc-LLVY-AMC (N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin) substrate (Enzo Life Sciences, Lörrach, Germany) 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 [62,63]. 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 FLUOstar Omega fluorometer (BMG Labtech, East Sussex, UK) 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 1 μM proteasome inhibitor MG132 or vehicle (DMSO).