Suc leu leu val tyr amc

Proteasome activity was evaluated using yeast proteasome (TUM Groll group). The inhibitory potency of the extracts against yeast proteasome was assessed by using the Fluorescence Intensity Assay and Suc-Leu-Leu-Val-Tyr-AMC as substrate (Enzo Life Sciences, BML-P802-0005). The assay was carried out at room temperature (22 °C) using Corning 4514 black low volume 384 well plates. All measurements were performed in singlicate and the final DMSO concentration was 3.3%. The assay buffer contained 100 mM Tris pH 7.5 and 1 mM MgCl2. First, 9.5 µL protein dilution (final concentration 9 nM) were mixed with 0.5 µL of either one of three probe dilutions (1, 0.1 or 0.01 mg/mL >> assay end conc. 0.033, 0.0033 or 0.00033 mg/mL) and preincubated for 90 min. Then 5 µL substrate mix were added (final concentration 5 µM) and incubated for 60 min. The assay plates were measured in fluorescence mode on a Tecan M1000 plate reader (ex 380 nm, em 460 nm); the IC50 was calculated using XLfit. ONX-0914, which inhibits yeast proteasome, was used as positive control in order to assess the functionality of the assay.

Dimethyl sulfoxide (DMSO), ethanol and 2-propanol were obtained from Fischer Scientific (Loughborough, UK). Naproxen, quercetin, sulphanilamide, N-(1-naphthyl)ethylenediamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS of Salmonella enterica, linoleic acid, LOX from glycine max (soybean) and trypan blue were obtained from Sigma-Aldrich (St. Louis, MO, USA). COX fluorescent inhibitor screening assay kit was purchased from Cayman chemicals (Ann Arbor, MI, USA). Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), heat inactivated foetal bovine serum (FBS), Pen-Strep solution (penicillin 5000 units mL⁻¹ and streptomycin 5000 mg mL⁻¹) and trypsin were obtained from Gibco Invitrogen TM (Grand Island, NY, USA). Proteasome 20S, lactacystin and Suc-Leu-Leu-Val-Tyr-AMC were purchased from Enzo Life Sciences. Proteasome 26S was obtained from AGS cells, as described below.

Proteasome peptidase assays were performed as previously described by us Aghdam et al. (2013 (link)). The cells were collected and lysed using the lysis buffer containing 50 mmol/L Tris‐HCl pH 7.4, 1 mmol/L ATP, 10% glycerol, 0.1% NP40, 2 mmol/L MgCl₂, 1.5 mmol/L DTT, and 0.03% SDS. BCA protein assay (Bio‐Rad) was used to determine protein concentrations. The protein lysates (50 μg/well) were added into a dark 96‐well microplate and probed with the fluorogenic peptide substrates: Z‐Leu‐Leu‐Glu‐AMC for caspase‐like, Ac‐Arg‐Leu‐Arg‐ AMC for trypsin‐like, and Suc‐Leu‐Leu‐Val‐Tyr‐AMC for chymotrypsin‐like (Enzo Life Sciences, Farmingdale, NY) at the final concentration of 100 μmol/L in a total volume of 0.1 mL. The plates were incubated in the dark for 30 min at 37°C. Following incubation, the reaction was stopped using 100% ethanol (100 μL/well). The fluorescent intensity was measured using a plate reader (PerkinElmer, Wellesley, MA). The excitation and emission wavelengths were 355 and 460 nm, respectively.

The detection method of proteasome activity is based on the principle of detecting fluorophore 7-amino-4 metholcoumarin (AMC) cleaved by 20S proteasome from the labelled substrate Leu-Leu-Val-Tyr-AMC [40 (link)]. Cells were washed with PBS for three times, and then lysed with analytical lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100; 2 mM ATP). The cell lysate was placed on ice for 30 min, vortex every 5 min, and then centrifuged at 12000 g for 10 min. The supernatant after centrifugation was collected and the protein concentration was determined by BCA kit. Ten microgram of cell proteins was diluted to 90 μl with analytical buffer (50 mM Tris HCl, pH 7.5; 150 mM NaCl). Then 10 μl of the synthetic fluorogenic substrates Suc-Leu-Leu-Val-Tyr-AMC (500 μM, Enzo Life Sciences) was added to each assay reaction (final concentration of 50 μM in a total volume of 100 μl). The samples were incubated at 37°C and the fluorescence signal was read in Molecular Devices (Flexstation 3) microplate detection system with excitation wavelength of 355 nm and emission wavelength of 460 nm.

Measurement of proteasome proteolytic activities was performed as previously described [22 (link),56 (link)]. Briefly, human fibroblasts were plated in 60-mm dishes and were treated for 24 h with a concentration of 5 μM for each compound and 1 μg/mL and 10 μg/mL of concentration for each extract. After the treatment cells were lysed with a buffer suitable for the isolation of 26S proteasome (0.2% Nonidet P-40, 5 mM ATP, 10% glycerol, 20 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and 20 mM Tris, pH 7.6). Lysates were cleared with centrifugation at 19,000g (4 °C) and 20 μg of proteins were immediately used to determine the main proteasome proteolytic activities [chymotrypsin-like (CT-L/LLVY) and caspase-like (C-L/LLE)]. Activities were assayed by recording the hydrolysis of the fluorogenic peptides Suc–Leu–Leu–Val–Tyr–AMC and Z-Leu–Leu–Glu–AMC (Enzo Life Sciences), at 37 °C for 30 min. The fluorescence was measured at a VersaFluorTM Fluorometer System (Bio-Rad laboratories) at excitation and emission wavelengths of 350 and 440 nm, respectively. Each sample was prepared in duplicate.