Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates
Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates
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DOI：
10.1016/s1357-2725(02)00391-6
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发表时间：
2003-05-01
影响因子：
4
通讯作者：
Dean, RT
中科院分区：
文献类型：
--
作者：
Rodgers, KJ;Dean, RT
泛素-蛋白酶体途径是细胞蛋白质降解的主要途径。它还通过降解许多限速酶和关键调节蛋白在维持细胞稳态中起重要作用。蛋白酶体活性的改变与许多病理学有关，包括帕金森病、阿尔茨海默病和糖尿病。真核生物蛋白酶体是一种多催化蛋白酶，具有三种活性，对肽底物具有不同的特异性。虽然底物被确定，可以选择性地测量纯化的蛋白酶体中的个体活性，但当与可能含有许多其他活性肽酶的生物样品一起使用时，关于这些底物对蛋白酶体活性的特异性如何的数据很少。在这里，我们研究了三个主要的肽酶活性的两种细胞类型的裂解物中，并在肝细胞质组分中的存在下，特定的蛋白酶体抑制剂，并通过凝胶渗透色谱分离后。我们证明，在这些制剂中存在的其他蛋白酶可以在标准测定条件下降解常用的蛋白酶体底物。我们开发了一种简单的方法，用于分离的蛋白酶体从较低分子量的蛋白酶使用500 kDa的分子量截止膜。这使得蛋白酶体活性可以在粗生物样品中准确测量，并且可以具有相当广泛的适用性。我们还确定了低分子量的胰蛋白酶活性的细胞和组织的制剂，不能被抑制的蛋白酶体抑制剂环氧霉素，但两个半胱氨酸蛋白酶抑制剂和lactacystin，这表明lactacystin可能不完全是蛋白酶体特异性的rectifitable。（C）2003 Elsevier Science Ltd.保留所有权利。
The ubiquitin-proteasome pathway is a major route of degradation of cell proteins. It also plays an essential role in maintaining cell homeostasis by degrading many rate-limiting enzymes and critical regulatory proteins. Alterations in proteasome activity have been implicated in a number of pathologies including Parkinson's disease, Alzheimer's disease and diabetes. The eukaryotic proteasome is a multicatalytic protease characterized by three activities with distinct specificities against peptide substrates. Although substrates were identified which could selectively measure the individual activities in the purified proteasome little data is available on how specific those substrates are for proteasomal activity when used with biological samples which may contain many other active peptidases. Here we examine the three major peptidase activities in lysates of two cell types and in a liver cytosol fraction in the presence of specific proteasome inhibitors and after fractionation by gel permeation chromatography. We demonstrate that other proteinases present in these preparations can degrade the commonly used proteasome substrates under the standard assay conditions. We develop a simple method for separating the proteasome from the lower molecular weight proteases using a 500 kDa molecular weight cut-off membrane. This allows proteasome activity to be accurately measured in crude biological samples and may have quite broad applicability. We also identify low molecular weight tryptic activity in both the cell and tissue preparations which could not be inhibited by the proteasome inhibitor epoxomycin but was inhibitable by two cysteine proteinase inhibitors and by lactacystin suggesting that lactacystin may not be completely proteasome specific. (C ) 2003 Elsevier Science Ltd. All rights reserved.