Richard Moravec, Martha O'Brien, Tracy Worzella, Bill Daily*, Mike Scurria*, Laurent Bernad*, Kay Rashka, Jeri Culp, Sandra Hagen, Brian McNamara, Alyssa TenHarmsel and Terry Riss
Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711
*Promega Biosciences Inc., 277 Granada Dr., San Luis Obispo, CA 93401
We have developed a series of cell-based assays that individually measure the chymotrypsin-like, trypsin-like or caspase-like activities of the proteasome. Each assay utilizes a specific luminogenic proteasome peptide substrate in a buffer optimized for cell permeabilization, proteasome activity and luciferase activity. Addition of the reagent directly to cultured cells gently permeabilizes cellular plasma membranes, allowing substrate and reagent access to the cytosolic proteasome. Luminescence is generated in a coupled enzyme format, as proteasome cleavage of the peptide substrate generates aminoluciferin, which is a substrate for luciferase. A stable glow-type luminescent signal is obtained 10-15 minutes after addition of reagent. Luminogenic proteasome substrates enabled development of the "add-mix-measure" assays with adequate sensitivity to be miniaturized into 96 and 384-well plate formats.