Optimal determination of heart tissue 26S-proteasome activity requires maximal stimulating ATP concentrations

The ubiquitin-proteasome system has been implicated in both cardiac physiology and pathophysiology. Research in this area has been hampered by the lack of a simple, reproducible method to assess 26S-proteasome peptidase activities. The current report demonstrates that one reason for lack of reproducibility is the myriad of ATP concentrations, many of them excessive, which have been used to stimulate peptidase activity. The chymotrypsin-like or caspase-like activities of 26S-proteasome in cardiac tissue isolates were determined using Suc-LLVY-AMC or Z-LLE-AMC, respectively, over a range of ATP concentrations up to 2 mmol/L. The optimal ATP concentration to assess both peptidase activities was found to be in the low micromolar range (from 6 to 100 μmol/L) depending on the cardiac tissue isolate protein (10 to 90 μg protein) contained in the reaction. Increasing ATP beyond the optimal range was inhibitory. In general, chymotrypsin-like and caspase-like activities could be stimulated 2- to 2.5-fold and 1.4- to 1.8-fold, respectively, over basal (ATP, 0 μmol/L), and could be effectively inhibited with lactacystin or Z-Pro-Nle-Asp-CHO, respectively. Based on these observations, an optimized method is presented for ex vivo determination of cardiac 26S-proteasome peptidase activities which was used to confirm inactivation of this complex by myocardial ischemia and reperfusion.