Boc leu arg arg amc

Chymotrypsin-like, trypsin-like, and caspase-like proteasome activities were
measured in mouse lung and mouse pulmonary artery after exposure to 3 weeks of
normoxia or hypoxia. Lung tissue from a single animal, or pulmonary arteries
pooled from four animals within the same treatment group, were prepared in
homogenization buffer (25 mM Tris pH 7.5, 100 mM NaCl, 5 mM ATP, 0.2% glycerol).
Samples were then centrifuged for 10 min at 5000 rpm at 4 ℃, the supernatant was
collected, and protein concentrations were determined by bicinchoninic acid
(BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL). To measure
proteasomal activity, samples were then incubated for 1 hour with assay buffer
[50 mM Tris pH 7.5, 2 mM dithiothreitol (DTT), 5 mM MgCl₂, 2 mM ATP]
containing the fluorogenic substrate [40 µM Suc-Leu-Leu-Val-Tyr-
7-amino-4-methylcoumarin (AMC), chymotrypsin-like; Boc-Leu-Arg-Arg-AMC,
trypsin-like; or Z-Leu-Leu-Glu-AMC, caspase-like; Enzo Life Sciences,
Farmingdale, NY]. Samples treated with either 1 µg purified human 20S Proteasome
(Enzo Life Sciences) or 40 nM MG132 were used as positive and negative controls,
respectively. Proteolytic activities were measured by observing the fluorescence
created by release of the AMC fluorescent group with a Wallac Victor 3
fluorimeter (PerkinElmer, Waltham, MA) every 10 minutes over 1 hour
(excitation = 390 nm and emission = 460 nm).

Cells were lysed on ice by using 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, after protein content adjustment with Bradford, supernatants were immediately used to determine the three proteasome proteolytic activities as described previously [14,25] . Briefly, the chymotrypsin-like (CT-L/LLVY), caspase-like (C-L/LLE) and trypsin-like (T-L/LRR) activities were assayed by recording the hydrolysis of the fluorogenic peptides Suc–Leu–Leu–Val–Tyr–AMC, Z-Leu–Leu–Glu–AMC, and Boc–Leu Arg–Arg–AMC (Enzo Life Sciences, Farmingdale, NY, USA), respectively, at 37 °C for 3 min. The fluorescence was measured at a VersaFluorTM Fluorometer System (Bio-Rad laboratories, Hercules, CA, USA) at excitation and emission wavelengths of 350 and 440 nm, respectively.