Limitations of SLLVY-AMC in calpain and proteasome measurements.
Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (SLLVY-AMC) is a fluorogenic substrate used to measure calpain activity and the "chymotrypsin-like" activity of the 20s proteasome. The goal of this study was to determine the relative role of calpains and the proteasome on SLLVY-AMC cleavage in attached and suspended renal epithelial cells (NRK-52E). The proteasome inhibitor epoxomicin did not inhibit purified calpain 1 or calpain 10 cleavage of SLLVY-AMC. Epoxomicin inhibited 11% of total SLLVY-AMC cleavage in attached cells and increasing concentrations of the calpain inhibitor calpeptin were additive. In contrast, cell suspensions had a 3.5-fold higher rate of SLLVY-AMC cleavage, epoxomicin inhibited cleavage 65% and calpeptin inhibited cleavage an additional 26%. Calpeptin alone also inhibited proteasomal activity. In conclusion, (1) SLLVY-AMC is cleaved in cells by calpain and the proteasome, (2) proteasome activity can be measured with epoxomicin, and (3) calpeptin can inhibit proteasome activity in some cases; thus limiting the use of SLLVY-AMC and calpeptin.
Giguere
Christopher J
CJ
Center for Cell Death, Injury, and Regeneration, Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina, Charleston, SC 29425-1400, USA.
Schnellmann
Rick G
RG
D000818:Animals; D002154:Calpain / Q000032:analysis* / Q000037:antagonists & inhibitors; D002460:Cell Line; D003374:Coumarins / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D061988:Proteasome Inhibitors; D011817:Rabbits; D051381:Rats
D003374:Coumarins; C114703:N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D002154:Calpain; D046988:Proteasome Endopeptidase Complex; C078846:epoxomicin