Optimal determination of heart tissue 26S-proteasome activity requires maximal stimulating ATP concentrations
Optimal determination of heart tissue 26S-proteasome activity requires maximal stimulating ATP concentrations
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DOI：
10.1016/j.yjmcc.2006.10.010
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发表时间：
2007-01-01
影响因子：
5
通讯作者：
Divald, Andras
中科院分区：
文献类型：
--
作者：
Powell, Saul R.;Davies, Kelvin J. A.;Divald, Andras
泛素-蛋白酶体系统与心脏生理学和病理生理学都有关系。这一领域的研究一直受到缺乏一个简单的，可重复的方法来评估26 S-蛋白酶体肽酶活性的阻碍。目前的报告表明，缺乏重现性的一个原因是大量的ATP浓度，其中许多浓度过高，这些浓度已被用来刺激肽酶活性。分别用Sue-LLVY-AMC或Z-LLE-AMC测定心肌组织分离物中26 S-蛋白酶体的糜蛋白酶样或半胱天冬酶样活性，ATP浓度范围为2 mmol/L。根据反应中所含的心脏组织分离蛋白（10 - 90 μ g蛋白），发现评估两种肽酶活性的最佳ATP浓度在低微摩尔范围内（6 - 100 μ mol/L）。增加ATP超过最佳范围是抑制性的。一般而言，胰凝乳蛋白酶样和半胱天冬酶样活性可分别刺激2- 2.5倍和1.4- 1.8倍，超过基础（ATP，0 μ mol/L），并可有效地抑制lactacystin或Z-Pro-Nle-Asp-CHO，分别。基于这些观察结果，提出了一种优化的方法，用于离体测定心脏26 S-蛋白酶体肽酶的活性，用于确认心肌缺血和再灌注的这种复合物的失活。(c)2006年爱思唯尔公司All rights reserved.
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 Sue-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 mu mol/L) depending on the cardiac tissue isolate protein (10 to 90 mu g 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 mu mol/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. (c) 2006 Elsevier Inc. All rights reserved.