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Updated: Jul 5, 2026
Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
Published on: July 8, 2010
Limitations of SLLVY-AMC in calpain and proteasome measurements
Christopher J Giguere1, Rick G Schnellmann
1Center 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.
Abstract:
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.
