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Comment
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Peptide-fluoromethyl ketone inhibitor of Caspases-1 and -4. The CH2F (fluoromethyl ketone) inhibitor has several advantages over other types of derivatives: Penetrates cell membranes, Not toxic to cells, Irreversible inhibition.
Interleukin-1ß Converting Enzyme (ICE), also now known as Caspase-1, is a cytoplasmic cysteine protease that cleaves inactive 31 kDa pro-IL-1ß to generate the active 17.5 kDa proinflammatory cytokine IL-1ß, the predominant form of IL-1 produced by human monocytes. This cytokine has been implicated in the pathogenesis of several diseases such as rheumatoid arthritis, inflammatory bowel disease, and septic shock.
Caspase-1/ICE mRNA is found in a variety of cells such as peripheral blood monocytes, peripheral blood lymphocytes, peripheral blood neutrophils, and resting and activated peripheral blood T-lymphocytes. The tissue distribution of Caspase-1/ICE suggests that the enzyme may have other substrates in addition to IL-1ß. Current hypotheses suggest that Caspase-1/ICE is able to cause apoptosis as well as activate inflammation in animal cells. Experiments have shown that Caspase-1/ICE has sequence homology with other mammalian apoptosis genes and that activation of Caspase-1/ICE or other caspase proteases is required for anti-Fas mAb-induced apoptosis.
Strong inhibition of Caspase-1/ICE and weak inhibition of Caspase-4. No inhibition of Caspases-2, -3, -6, or -7.
Dissolve the Caspase-1/ICE Inhibitor 2 in high purity (>99.9%) DMSO before use to make a stock solution of 20 mM.
For use on intact cells:
1. Prepare stock solutions as follows:
5 mg Z-YVAD-FMK in 794 µl DMSO = 10 mM
3 mg Z-YVAD-FMK in 476 µl DMSO = 10 mM
1 mg Z-YVAD-FMK in 159 µl DMSO = 10 mM
2. Add 2 µL of 10 mM stock solution to 1 mL culture medium containing cells such that the fµl DMSO concentration is 0.2%. Levels of DMSO above this may cause some cellular toxicity, thus masking the effect of the ICE- protease inhibitors. Adding 2 µL of a 10 mM stock solution to 1 mL of culture medium gives a final Z-YVAD-FMK concentration of 20 µM. Effective final concentrations are estimated to be 5-20 µM.
For extended use in vivo or in vitro:
For experiments extending 12 to 48 hours, fresh inhibitor may have to be added (injected) due to inactivation of the inhibitor by endogenous cysteine proteases.
IMPORTANT NOTE for in vitro use: Our peptide inhibitors are synthesized as methyl esters to enhance cell permeability. In intact cells, the methyl groups are removed by endogenous enzymes. For in vitro experiments with purified enzymes, however, the methyl groups must first be removed by treating the inhibitor with esterase. A procedure is available upon request.
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