Limitations of SLLVY-AMC in calpain and proteasome measurements

Christopher J. Giguere, Rick G. Schnellmann

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)578-581
Number of pages4
JournalBiochemical and Biophysical Research Communications
Volume371
Issue number3
DOIs
StatePublished - Jul 4 2008
Externally publishedYes

Keywords

  • Calpain
  • Calpeptin
  • Epoxomicin
  • Proteasome
  • SLLVY-AMC

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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