Fluorometric and colorimetric detection of caspase activity associated with apoptosis.
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.
Gurtu
V
V
CLONTECH Laboratories, Inc., Palo Alto, California 94303, USA.
Kain
S R
SR
Zhang
G
G
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003124:Colorimetry / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D009842:Oligopeptides; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases