Fluorometric and colorimetric detection of caspase activity associated with apoptosis
Fluorometric and colorimetric detection of caspase activity associated with apoptosis
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DOI：
10.1006/abio.1997.2220
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发表时间：
1997-08-15
影响因子：
2.9
通讯作者：
Zhang, GH
中科院分区：
文献类型：
--
作者：
Gurtu, V;Kain, SR;Zhang, GH
The caspase (ICE/CED-3) family of proteases has been implicated to play a crucial role in apoptosis. However, the mechanisms by which caspase activity mediates apoptosis are not fully understood. Progress in this area has been limited due to the lack of a convenient and reliable system to quantify these protease activities. In this report, we describe a quantitative assay for the activity of caspase-3, a member of the caspase family thought to mediate apoptosis in most mammalian cell types. This assay utilizes a synthetic tetrapeptide, Asp-Glu-Val-Asp (DEVD), labeled with either a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC), or a colorimetric molecule,p-nitroanilide (pNA) as substrates. DEVD-dependent protease activity is assessed by detection of the free AFC or PNA cleaved from the substrates. We demonstrate the utility of the assay for rapid quantification of caspase-3 activity in the onset of apoptosis. Using the assay, we show that apoptosis induced in 32D cells under various conditions is associated with an increase in the DEVD-dependent protease activity, These studies suggest that induction of the DEVD-dependent protease activity is an indicator of apoptosis and demonstrate the utility of the assays for assessment of the role of caspase-family proteases in apoptotic cell progression. (C) 1997 Academic Press.
