2.5. Proteasome activity

The rate of degradation of fluorogenically labeled suc-LLVY-AMC was measured for determining the chymotrypsin-like activity of the β − 5 catalytic subunit located in the 20S core particle [1]. Cells were cultured in 6-well (34.8 mm diameter) plates and treated when reached 80% confluency. After treatment cells were trypsinized, placed in 1.5 ml tubes and washed three times by subsequent centrifugation (2000 ×g at 4 °C) and resuspension in ice-cold PBS. Washed cell pellets were resuspended in proteasome lysis buffer (25 mM HEPES, 250 mM sucrose, 20 mM MgCl₂, 1 mM EDTA, pH 7.4), lysed by three freeze-thaw cycles (liquid nitrogen and 37 °C water bath, respectively). Cell lysates were centrifuged at 14,000 ×g for 30´ at 4 °C to remove cell debris. The supernatants were placed in new tubes and the protein content was measured by the DC Protein Assay Lowry. All samples were diluted to a concentration of 1 µg/µl. 10 µl (10 µg) of sample (triplicates) were placed in a black 96-well plate. 90 µl of proteasome activity buffer (150 mM Tris, 30 mM potassium chloride, 7.5 mM MgOAc, 10 mM MgCl₂, containing either 100 µM ATP for measuring the ATP- stimulated proteolysis, or 15 mM deoxyglucose and 0.1 mg/ml hexokinase for depletion of ATP) was added and incubated during 20´ at RT. The reaction was started by addition of 20 µl of 120 µM suc-LLVY-AMC diluted either in ATP-containing or ATP-depletion buffer. Methyl coumarin liberation was measured with a fluorescence plate reader (λ_ex= 360 nm, λ_em= 485 nm). The chymotrypsin-like activity of the proteasome was determined as the rate of methyl coumarin fluorescence increase during the first 30 min of reaction.