Tissue homogenates containing proteasome were prepared just after dissection using ice-cold homogenization buffer containing: 20 mM Tris-HCl (pH 7.6), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA and 2 mM DTT (Hovhannisyan et al., 2019 (link)). Samples were minced with scissors and homogenized using plastic pestles, incubated 30 min on ice, then centrifuged at 12,000 g for 15 min at 4°C. Protein concentration was measured using the Bradford method with bovine serum albumin as a standard. The proteasomal chymotrypsin-like, trypsin-like and caspase-like activities of the 20S catalytic core were assayed using the fluorogenic substrates N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC, Enzo Life Sciences), Bz-Val-Gly-7-amino-4-methylcoumarin (Bz-VGR-AMC, Enzo Life Sciences) and Z-Leu-Leu-Glu-7-amino-4-methylcoumarin (Z-LLE-AMC, Enzo Life Sciences), respectively. The mixture, containing 10 μg of total protein in 20 mM Tris (pH 8) and 10% gylcerol, was incubated at 37°C with 20 μM peptide substrates in a final volume of 100 μl. Enzymatic kinetics were monitored in a temperature-controlled microplate fluorimetric reader (FLUOstar Galaxy, BMG labtech). Excitation/emission wavelengths were 350/440 nm. The difference between assays with or without MG-132, a proteasome inhibitor, represented the proteasome-specific activity.
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