Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells
Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells
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DOI：
10.1038/labinvest.3700340
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发表时间：
2005-11-01
影响因子：
5
通讯作者：
Takata, M
中科院分区：
文献类型：
--
作者：
Alvarez-Iglesias, M;Wayne, G;Takata, M
肿瘤坏死因子- α (TNF)转换酶（TACE）负责多种膜蛋白的脱落，包括促炎细胞因子TNF。由于缺乏可靠的方法来测量基于细胞的TACE活性，人们对TACE的体内调节知之甚少。在这里，我们报告了一种新的酶分析，可以连续实时测量活细胞表面的TACE活性。用一种新的荧光共振能量转移肽（由tace敏感的TNF序列和荧光素-四甲基罗丹明（FAM-TAMRA）组成）培养细胞，并通过肽切割引起的荧光信号增加率来监测酶活性。使用静息和刺激单核细胞的验证研究表明，该分析是敏感的，可重复性和定量的。各种抑制剂的药理学研究表明，观察到的酶活性可能主要归因于TACE。因此，FAM - TAMRA肽为测量组成型和诱导型细胞TACE活性提供了一个强有力的工具。所建立的原理可应用于活细胞上各种酶活性的分析。
Tumor necrosis factor-alpha (TNF) converting enzyme (TACE) is responsible for shedding of various membrane proteins including proinflammatory cytokine TNF. In vivo regulation of TACE is poorly understood mainly due to lack of reliable methodology to measure TACE activity in cell-based assays. Here we report a novel enzyme assay that enables continuous real-time measurement of TACE activity on the surface of live cells. Cells were incubated with a new fluorescent resonance energy transfer peptide consisting of a TACE-sensitive TNF sequence and fluorescein - tetramethylrhodamine (FAM-TAMRA), and enzyme activity was monitored by the rate of increase in fluorescent signal due to peptide cleavage. Validation studies using resting as well as stimulated monocytic cells indicated that the assay was sensitive, reproducible and quantitative. Pharmacological studies with various inhibitors indicated that the observed enzyme activity could largely be ascribed to TACE. Thus, the FAM - TAMRA peptide provides a powerful tool for measurement of constitutive and inducible cellular TACE activity. The principles developed may be applied to analyses of enzyme activity of various sheddases on live cells.