The proteasome is a large, multi-catalytic protease complex within the eukaryotic cell. The 26S proteasome, a 60-subunit particle, is composed of a 20S core and one or two 19S regulatory particles. The catalytic core known as the 20S proteasome (700 kDa) is composed of two sets of seven β-subunits and two sets of seven α-subunits. The β1, β2, and β5 subunits possess caspase-like, trypsin-like, and chymotrypsin-like peptidolytic activities, respectively. In eukaryotic cells, the 20S proteasome is assembled with the aid of at least five assembly chaperones. The degradation of cellular proteins by the proteasome is initiated by attachment of an ubiquitin chain to the polypeptide. The ubiquitin tag is recognized by and binds to the ‘lid’, a 19S particle, followed by disassembly of ubiquitin chains, protein unfolding and subsequent translocation into the 20S proteasome.
Proteasomal activity plays essential role in major cellular events including protein quality control, regulation of gene transcription, DNA damage response, aging, oxidative stress and signal transduction. Many selective proteasome inhibitors targeting specific active sites have been identified in the last two decades. Most notably, Bortezomib and Carfilzomib are FDA-approved proteasome inhibitor drugs for the treatment of multiple myeloma. Several new proteasome inhibitors are currently in trials for the treatment of multiple myeloma and other malignancies.
This purified 20S proteasome preparation can be used in vitro for the degradation of peptide substrates and unfolded proteins. Chymotrypsin-like activity of the proteasome is readily measured using Suc-LLVY-AMC (Cat. # PS500). Upon cleavage by the active enzyme, it generates a highly fluorescent product with an emission wavelength at 460 nm.
- The chymotrypsin-like activity of the 20S proteasome (45µg/ml) was measured using 100 µM Suc-LLVY-AMC in 20 mM HEPES, pH 7.5, 0.5 mM EDTA, 0.05% Triton X, plus or minus 0.035% SDS.
- The chymotrypsin-like activity of the 20S proteasome (18 µg/ml) was measured using 100 µM Suc-LLVY-AMC in 20 mM HEPEs, pH 7.5, 0.5 mM EDTA, 0.05% Triton X, plus or minus 0.5 mM MG132 (Cat. # SI9710).
|Source||Red blood cells|
|Molecular Weight||700 kDa|
|Formulation||20 mM Tris-HCl pH 7.2, 10% Glycerol, 150 mM KCl, 1 mM β– mercaptoethanol|
|Storage||-80°C, avoid freeze/thaw cycles|