Detection of functional matrix metalloproteinases by zymography.
Detection of functional matrix metalloproteinases by zymography.
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DOI：
10.3791/2445
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发表时间：
2010-11-08
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影响因子：
--
通讯作者：
Beeton, Christine
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文献类型：
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作者：
Hu, Xueyou;Beeton, Christine
基质金属蛋白酶(MMPs)是一种含锌的内肽酶。它们通过裂解多肽键来降解蛋白质。目前已鉴定出20多种MMPs，并根据其结构和底物特异性将其分为六类(胶原酶、明胶酶、膜型[MT-MMPs]、基质蛋白、基质蛋白等)。MMPs在细胞侵袭、软骨降解、组织重塑、伤口愈合和胚胎发生等过程中发挥重要作用。因此，它们既参与正常的过程，也参与许多疾病的发病，如类风湿性关节炎、癌症或慢性阻塞性肺疾病。在这里，我们将重点介绍一种广泛表达的明胶酶A、IV型胶原酶-2。我们将演示如何通过酶图谱检测细胞培养上清液中的基质金属蛋白酶-2，这是一种常用、简单但非常敏感的技术，首先由C.Heussen和E.B.Dowdle于1980年提出。这项技术是半定量的，因此，当已知浓度的重组基质金属蛋白酶被加载到同一凝胶上时，它可以用于测定测试样品中的基质金属蛋白酶水平。含有MMPs的溶液(如细胞培养上清液、尿液或血清)被装载到含有十二烷基硫酸钠(十二烷基硫酸钠；以线性化蛋白质)和明胶(基质金属蛋白酶-2的底物)的聚丙烯酰胺凝胶上。样品缓冲液的设计目的是增加样品粘度(便于凝胶加载)，提供跟踪染料(溴酚蓝；监测样品迁移)，提供变性分子(使蛋白质线性化)，并控制样品的pH值。然后，允许蛋白质在电流作用下在运行缓冲器中迁移，该缓冲器旨在提供恒定的迁移速率。迁移距离与蛋白质的分子量成反比(小的蛋白质在凝胶中移动的速度比大的蛋白质快，因此在凝胶中迁移得更远)。迁移后，凝胶被放置在复性缓冲液中，以允许蛋白质重新获得酶活性所必需的三级结构。然后将凝胶放入显影缓冲液中，该缓冲液旨在允许蛋白酶消化其底物。显影缓冲液还含有对氨基苯基汞醋酸酯(APMA)，用于将非蛋白水解性的前MMPs激活为活性MMPs。下一步是对底物(在我们的例子中是明胶)进行染色。在将多余的染料从凝胶上洗掉后，蛋白酶消化区显示为清晰的条带。条带越清晰，它所含的蛋白酶就越集中。然后，可以使用ImageJ等软件通过密度测定来确定条带染色强度，从而允许进行样本比较。
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel. Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.