4.16. Proteasomal Activity Assays

The proteasome activity was determined with fluorogenic peptides Z-ARR-AMC (trypsin-like activity); Suc-LLVY-AMC (chymotrypsin-like activity); and Z-LLE-AMC (caspase-like activity). Briefly, cells were centrifuged, washed twice with cold sterile water, and suspended in protein extraction buffer (50 mM HEPES buffer with pH 7.8, containing 10 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, and 5 mM DTT). The cells were disrupted with 0.5 mm glass beads in 7 cycles of 30 s with intervals for cooling the sample in ice and centrifuged (14,000 g, 15 min, 4 °C). The degradation of fluorogenic peptide was measured by continuously monitoring the fluorescence of the reaction product, free 7-amino-4-methylcoumarin (AMC). The assay was performed in a protein extraction buffer supplemented with 2 mM ATP. The rate of fluorescence increase was measured using a TECAN Infinite 200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at 37 °C for 60 min at λ_ex = 350 nm and λ_em = 440 nm. The values were expressed in arbitrary units.