Caspases plate

Figure 7.4 Activation of PARP-1 by DNA breaks. PARP-1 is composed of three domains, DNA binding, automodification and catalytic (NAD+ binding) domains (1). In cells, PARP-1 localizes to nucleoli and actively transcribed regions of chromatin by interacting with RNA. When PARP-1 binds to DNA breaks, PARP-1 initiates the poly(ADP-ribosyl)ation reaction by using NAD+ as its substrate (2). PARP-1 itself is the main target of the poly(ADP-ribosyl)ation reaction. ADP-ribose polymers are formed on the automodification domain of PARP-1 (automodification). As a consequence of automodification, PARP-1 dissociates from DNA breaks (3). When cells are committed to apoptosis, PARP-1 is specifically cleaved by an apoptosisspecilic protease, caspase-3, resulting in the formation of a 24kDa N-terminal and 89 kDa C-terminal fragments (4). (see Color Plate 7)...

Caspases are among the most specific proteases known, Thanks to the crystal structures of active caspases (caspase 1 and 3), we have some idea how these enzymes are activated. The structure of the interleukin-ip-convertii enzyme (ICE), which processes an inactive interleukin precursor to interleukin-ip, is shown in Plate 26. 

The active caspase ELISAs quantify the events closely following the release of cytochrome c from mitochondria. The active caspase ELISA quantifies a single active caspase family member (19). The procedure uses the covalent modification of the active site cysteine with a biotinylated inhibitor to label active caspases and selected capture on a plate coated with a caspase-specific monoclonal antibody (MAb). In this chapter, a typical characterization of an active caspase ELISA is included as a demonstration of proof that the ELISA quantifies a specific active caspase. The ability of the assay to... 

Results typical of those obtained when mitochondria, HRP-conjugated detection antibody, and Bcl-2 family members were incubated in assay buffer in wells of the ELISA plate for 30 min at 30°C are shown in Fig. 4A. Caspase-8 cleaved human Bid was used to demonstrate that the short format detects protein-induced cytochrome c release (Fig. 4A). Bcl-xL was added with cleaved Bid to demonstrate that the short format detects inhibition of a pro-apoptotic Bcl-2 family member by an anti-apoptotic member (Fig. 4A). In the 35-min assay, Bid-induced release of cytochrome c was concentration-dependent with an EC50 of 38 nM (Fig. 4B). The difference between the EC50 of 38 nM determined in the 35-min short-format assay was not statistically different (z test, p < 0.05) from the EC50 of 30 +/- 4 nM determined in the 3-h long format assay (Fig. 3A). Appreciable release caused by cleaved Bid can also be detected in a 15-min short-format assay conducted in the well of the ELISA plate (Fig. 4C) and inhibition of Bid was apparent when Bcl-xL was included in the 15-min short-format assay (Fig. 4C). 

Fig. 4. Detection of cytochrome c release from mitochondria in the short format cytochrome c release assay. (A) Assay buffer, HRP-detection antibody, and 52 nM caspase-8-cleaved human Bid (solid bars), 52 nM human cleaved Bid and 156 nM mouse Bcl-xL (open bars), no additions (light gray bars), or Triton X-100 (stippled bars), were added to a 96-well ELISA plate coated with cytochrome c capture antibody. Mitochondria were added and the plate was incubated at 30°C for 30 min (A) or 10 min (C). The wells were washed for 2 min and color developing reagent was added for 3 min. (B) shows a 35-min assay in which the concentration of human cleaved Bid was varied. Open triangle indicates cytochrome c detected when Triton X-100 was added to mitochondria and open diamond indicates cytochrome c detected when no Bcl-2 family proteins were added to mitochondria.

Specificity of the active caspase-7 ELISA was determined by analyzing captured polypeptides. Capture antibody is the caspase-7-specific antibody coated in the ELISA 96-well plates and is different from the anti-caspase-7 antibodies used for western blotting. Polypeptides that are bound by the capture antibody are referred to as captured. Jurkat cells were incubated with STS for 0-4 h and then with bzVKD-fmk for an additional 1 h. Cell extracts were incubated in 6-well plates coated with caspase-7 capture antibody. After washing, polypeptides captured on the plate were solubilized in SDS sample buffer and Western-blotted. Captured polypeptides were blotted with anti-caspase-7 LSU and anti-caspase-7 SSU to detect polypeptides derived from caspase-7. Captured polypeptides were blotted with HRP-streptavidin to detect the polypeptides covalently modified with the bzVKD-fmk inhibitor. Captured polypeptides covalently modified with bzVKD-fmk is the material that the ELISA quantifies. 

We have characterized the activities of several recombinant human and mouse Bcl-2 family proteins (Bcl-2, Bcl-xL, Bcl-w, Bax, Bid, and caspase-8 cleaved Bid) with the cytochrome c ELISA. Dose responses, kinetics, inhibition, and the effects of protein-protein interactions have been quantified. Conducting the cytochrome c release assay in the well of the ELISA plate decreases the time required to perform the assay from 3 h to 35 min. Similar dose responses for induction of cytochrome c release caused by cleaved Bid were obtained with the 35 min and 3 h release assays, indicating that the short format can detect intermediate levels of cytochrome c release. The short format is applicable to automation for screening million-member libraries. Mitochondria obtained from one mouse liver is sufficient to perform 4000 assays in the 96-well plates and scale-up is performed simply by increasing the amount of starting tissue. 

To identify the biotinylated proteins that were captured by the caspase-7 capture antibody coated on the plates, 3 x 107 Jurkat cells were incubated for the indicated times with 1 iM STS. bzVKD-fmk was then added to the medium to give a final concentration of 10 [iM. After 1 h, cells were washed with PBS, solubilized in 1 mL of extraction buffer (extraction buffer and diluents were made as described in or provided with the active caspase kits) containing 6 M urea and protease inhibitors and stored overnight at 4°C. Extracts were diluted with 4 mL ELISA calibrator diluent and incubated for 2 h at room temperature in a 6-well dish coated with caspase-7 capture antibody. Captured material was washed 4 times with ELISA wash buffer and then 1 time with PBS. Captured proteins were solubilized directly into 200 uL SDS sample buffer. Captured proteins were separated by SDS-PAGE, and transferred to immobilon membranes (Millipore) that were subsequently incubated with HRP-streptavidin or anti-caspases-7 LSU or anti-caspase-7 SSU. 

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