Optimal determination of heart tissue 26S-proteasome activity requires maximal stimulating ATP concentrations - PubMed (original) (raw)

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

Saul R Powell et al. J Mol Cell Cardiol. 2007 Jan.

Abstract

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 micromol/L) depending on the cardiac tissue isolate protein (10 to 90 microg 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 micromol/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.

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Figures

Figure 1

Figure 1. Optimal conditions for determination of cardiac 26S-proteasome activity. Panel A: The effect of ATP concentration

Aliquots (30 μg protein) of rat heart tissue cytosol was assayed for chymotrypsin-like and caspase-like activities in the presence of increasing concentrations of ATP. The inset shows the data normalized to the 0 μmol/L ATP or basal activity to determine magnitude of activation. The values represent the mean ± SEM of 5 to 7 determinations. Panel B: The effect of increasing protein concentrations. Aliquots of rat heart tissue cytosol containing up to 90 μg protein were assayed for chymotrypsin-like and caspase-like activities in the presence of increasing concentrations of ATP. The values represent the mean of 2 to 3 determinations. Panel C: The effect of proteasome inhibitors. Aliquots (30 μg protein) of rat heart tissue cytosol was assayed for chymotrypsin-like and caspase-like activities at the optimal ATP concentration of 28 μmol/L and in the presence of increasing concentrations of the proteasome inhibitors, lactacystin (Lac) and Z-Pro-Nle-Asp-CHO (ZPNAC). The inset shows the effect of increasing ATP concentrations on chymotrypsin-like activity in the absence and presence of Lac, 10 μmol/L. The values represent the mean of 2 to 3 determinations.

Figure 2

Figure 2. Inhibition of 26S-Proteasome peptidase activity by ischemia and reperfusion

Isolated rat hearts were subjected to 30 min normothermic global ischemia followed by 60 min reperfusion. Hearts were harvested and analyzed for chymotrypsin-like and caspase-like activities. The values represent the mean ± SEM of 3 separate experiments and are expressed as raw data (left panels) and normalized to the 0 μmol/L ATP point (right panels).

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