Free amc

Portions of frozen livers were processed and then assayed for specific caspase activities following manufacturers’ instructions using commercially available caspase-3 fluorogenic substrate, Ac-DEVD-AMC (BD Biosciences, San Diego, CA, USA). Caspase activity was evaluated by measuring the release of AMC (7-Amino-4-methylcoumarin) obtained by the cleavage of the defined synthetic peptide sequence by caspase-3, using a Perkin Elmer Luminescence Spectrophotometer LS 50B. Free AMC obtained from Sigma-Aldrich (St Louis, MO, USA) was used as the standard. Protein was determined using the BCA Protein Assay kit from Pierce. The caspase-3 activity was expressed as pmoles of AMC produced per mg liver protein.

Proteasome chymotryptic and caspase activity was assayed as the rate of hydrolysis of the fluorogenic peptide suc-LLVY-AMC (Sigma Aldrich) or Z-LLE-AMC (Enzolifesciences, Farmingdale, New York), respectively. Cell extracts were prepared in 25 mM Tris HCl, pH 7.5 using sonication. Protein lysate (20 µg) was incubated with 12.5 µM suc-LLVY-AMC or Z-LLE-AMC in a total volume of 200 µl. MG132 was spiked into the reaction well at a final concentration of 0.1 µM where indicated. AMC fluorescence was measured using 355 nm excitation and 460 nm emission filters with free AMC (Sigma Aldrich) as standard every min for 30 min at 37°C.