20S Proteasome Assay Kit, SDS-Activation Format

The Ubiquitin Proteasome Pathway (UPP) is the cell’s principle mechanism for protein catabolism. The UPP has been shown to have significant involvement in the regulation of critical cellular processes such as transcription, oncogenesis, cell cycle progression, development, growth, selective elimination of abnormal proteins, and antigen processing (1-3). The proteasome is a large, multimeric protease that catalyzes the final step of the UPP intracellular protein degradation. The proteasome exists in multiple forms within the eukaryotic cell, and contained in all isoforms is the catalytic core known as the 20S proteasome. The 20S proteasome (700 kDa) is arranged as four axially stacked heptameric rings with two β-subunit rings sandwiched between two α-subunit rings. The multicatalytic centers are located within the internal cavity of the β-subunits. The 20S proteasome is characterized by three distinct proteolytic activities against short synthetic peptides: chymotryptic-like (Tyr or Phe at P1), tryptic-like (Arg or Lys at P1) and peptidylglutamyl peptide- hydrolyzing (Glu at P1) (4). These activities are believed to be catalyzed by the nucleophilic N-terminal threonines of the β-subunits (5, 6). The 20S proteasome in vitro cannot efficiently degrade peptides or proteins unless they are highly denatured and the 20S has been activated by the addition of low concentrations of sodium dodecyl sulfate (SDS) or PA28 (or 11 S activator) (7-12). The exact mechanism for this apparent SDS activation is not well-understood, except to postulate that SDS induces conformational changes under limited denaturation thereby allowing access to the central cavity where the active sites residues reside.

The most common assessment of 20S activity in vitro is done by measuring the hydrolysis of the fluorogenic peptidyl substrate Suc-Leu-Leu-Val-Tyr-AMC (Cat# S-280) by the SDS-activated proteasome. This substrate is cleaved by the chymotryptic-like activity of the proteasome releasing free AMC (7-amino-4-methylcoumarin) which can be efficiently detected using a fluorimeter (Ex_380nm; Em_460nm). Purified 20S proteasome chymotryptic-like activity is dependent on SDS concentration up to 0.030% SDS where the activity is maximal (Fig.1). SDS concentrations beyond 0.030% result in inactivation due to excessive and irreversible denaturation of the 20S. When this substrate is titrated against a fixed concentration of proteasome pre-activated with SDS, Suc-LLVY-AMC exhibits a KM value of 9 μM (Fig. 2). At saturating substrate concentrations (≥ 50 μM), the turn-over rate (V_max) is approximately 12 s-1.