Caspase Assay

Okamoto et al. (29) pursued development of peptidomimetic caspase-1 inhibitors based on the crystal structure of caspase-1/ Ac-Tyr-Val-Ala-Asp-H complex, which resulted in an inhibitor displaying potent activity in an in vitro caspase assay (IC50 =... 

Buhlmann C, Valer M, Mueller O (2004) Combination of DNA laddering and annexin-V and caspase assays on one system - multiple apoptosis parameters analysis with a microfluidic chip-based system. Tissue Antigens 64 392-392... 

Numerous bioassays format have been designed using derivatives of the mono-functionalized europium cryptate such as 47 (commercially available from CISbio, see http //www.htrf.com). Since the last reviews [127] some new applications Eu " C [BP.BP.BP] cryptate were developed through the HTRF technology, as caspase assay [128], telomerase assay [129], leukotriene b4 assay [130], inositol-1-phosphate assay [131], minisequencing [132], and MutS-DNA interaction [133]. Recently, the HTRF technology has been used in the study of protein-protein interactions and the authors demonstrated that measured values compare favorably with those calculated from independent experiments [134]. 

Rassi et al., 2002 Porcine bone marrow cells measure osteoclast formation (TRAP staining) and activity (pit assay) Daidzein, at the same concentration as estradiol, inhibits osteoclast formation and activity via caspase-3. 

Liu J, Bhalgat M, Zhang C et al (1999) Fluorescent molecular probes V a sensitive caspase-3 substrate for fluorometric assays. Bioorg Med Chem Letts 9 3231-3236... 

Karvinen, J., Laitala, V., Makinen, M. L., Mulari, O., Tamminen, J., Hermonen, J., Hurskainen, P. and Hemmila, I. (2004). Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal. Chem. 76, 1429-1436. 

Several biological assays pointed to an unusual mechanism of action for 7, which is clearly different from cisplatin, as expected from its remarkable cytotoxic activity. For example, different levels of p53 and caspase activity were observed. Unusual... 

The assay involves a chromogenic tag such as pNA linked to a specific peptide corresponding to the cleavage site of the respective caspase. Proteolysis of this peptide releases the chromogenic tag, thereby increasing the chromophore intensity. 

Apoptosis or programmed cell death is one of the regulatory mechanisms for the removal of unwanted cells. Apoptosis is induced by the stimulation of several different cell surface receptors in association with caspase activation. Apoptosis of a cell is thus a complicated process and can be assayed by various methods. Among widely used methods, the TUNEL assay is described here. 

Recently, a SAMDI-MS assay was described by means of which endogenous caspase protease activities in cell lysates can be determined [26], Similar to the assay used to determine anthrax lethal factor inhibitors, peptide substrate SAMs for either caspase-3 or -8 were treated with cell lysates. In contrast to fluorescence assays, also longer peptide substrates could be used, thus enabling a better resolution of the two caspase activities. 

Su, J., Rajapasksha, T.W., Peter, M.E., Mrksich, M. Assays of endogenous caspase activities a comparison of mass spectrometry and fluorescence formats. Anal. Chem. 2006, 78, 4945-4951. 

As with caspase-3, these hits were converted into potent, soluble inhibitors by replacing the disulfide linkage with a simple alkyl linkage (Fig. 9.10). As in the case of caspase-3, rigidifying the linker could improve affinity, as could introducing a hydrophobic moiety at the S2 position. Several of these molecules demonstrated activity in cellular assays and selectivity for caspase-1 over the closely related caspase-5. Crystallography of several of these molecules in complex with caspase-1 revealed that they bind in an extended conformation as expected, but that the S2 pocket that collapses in caspase-3 remains open in caspase-1. 

Fig. 12. Effect of spontaneous apoptosis on the activation of caspase-3 in human eosinophils. Eosinophils (2 x 10 /ml) were cultured for 3 h. Cells were lysed and caspase-3 activity was measured by caspase-3 colorimetric assay kit (R D Systems). Enzymatic products were measured at 405 nm with BIOTEK EL340 ELISA microplate reader (BIO-TEK Instrument Inc., Vermont). Human recombinant caspase-3 (5 U) (Calbiochem, California) was used as a positive control. The stimulation index was determined by direct comparison to the level of the normal control. Background readings from cell lysates and buffers were substracted from the readings of both induced and uninduced samples samples before calculating the stimulation index in caspase-3 activity. The differences between control and treated groups were assessed by Student s i-test. P < 0.05 P < 0.001 (Z2).

The EITC-VAD-FMK in situ assay is based on the use of EITC-VAD-EMK in situ marker (Promega Corp., Wisconsin) which is an EITC conjugate of the caspase inhibitor Z-Val-Ala-Asp (OMej-CHaF (Z-VAD-EMK) which irreversibly inhibits caspase-1, -3, -4, and -7. EMK allows delivery of the inhibitor into the cell where it irreversibly binds to activated caspases (E3). The EITC label allows for a direct, one-step assay of caspase activity in living cells or tissue section. The inhibitor... 

Activation of caspases 1.2 Colorimetric enzyme assay of caspase activity... 

A3. Ambroziak, J., Apoalert ELICE/caspase-8 fluorescent assay kit. Clontechniques 8, 6-7 (1998). 

Fig. 8.22. A diagram of some of the events associated with apoptosis, showing related flow cytometric assays (outlined in bold boxes). Although it is generally agreed that calcium flux and caspase activation are early events following the trigger signal, the events following on from there may be essential steps in an ordered pathway or may be independent and merely symptomatic of apoptosis.

In mammalian cells, there are multiple forms of enzymes within the same family, e.g., protein kinase C, phospholipase C, and caspase. A selective assay system would be instrumental in such cases to advance understanding of the respective role for each form within the same enzyme family. An example for design of a selective assay system for the superfamily of phospholipase A2 is provided in this chapter. Such a selective assay system may play a signihcant enabling role for PLA2 and other enzyme families in the discovery of inhibitors relevant to the treatment of pathologies involving those enzymes. 

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