Caspase-3/7

Ac-DEVD-AMC Fluorescent Labeling Protocol[2]:
Preparation of caspase buffer:
• 0.5% Igepal CA-630
• 10 mM HEPES (pH 7.4)
• 2 mM EDTA
• 0.5 mM PMSF
• 5 μg/mL leupeptin

Experimental procedures:
(1) PC12 cell treatment: 50 μM 6-OHDA for 20 hours
(2) Cell collection:
• Centrifuge at 250g for 5 minutes
• Wash with PBS
• Centrifuge again at 250g for 5 minutes
(3) Cell lysis:
• Resuspend cells in caspase buffer at 2μL/10⁵ cells
• Lyse at 4℃ for 20 minutes
(4) Lysate collection: Centrifuge at 7200g, 4℃ for 10 minutes
(5) Enzyme reaction:
• Incubate 20μL lysate with 100μM Ac-DEVD-AMC
• Incubate at 30℃ for 10 minutes
(6) Detection:
• Measure with fluorescence spectrophotometer
• Excitation wavelength: 380nm
• Emission wavelength: 460nm
Note: All centrifugation steps should be performed at 4℃.

Assaying CD95 (Fas/APO-1)-Activated Cytosolic Caspases. [1]
Jurkat cells [cultured as described in Nobel et al.], were exposed to 250−500 ng mL-1 anti-CD95 antibody for 1 h. Cytosolic S100 extracts were then prepared with a final protein concentration of 1.1−1.5 mg mL-1 as described; the extract contained approximately 200 μM GSH and 2 μM GSSG as determined by dansyl chloride derivatization and HPLC separation. For the enzyme assay 1 volume of S100 extract was mixed with 5 volumes of reaction buffer [100 mM Hepes, pH 7.25/10% (w/v) sucrose/0.1% (w/v) CHAPS/0.1 mM EDTA] containing the synthetic fluorogenic substrate Ac-DEVD-AMC (60 μM). Proteolytic activity against Ac-DEVD-AMC was assayed with a Fluoroskan II plate reader using AMC as a standard in a total volume of 60 μL.

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Enzyme Preparation and Enzyme Kinetics. [1]
Purified caspase-1 was provided as a mixed disulfide with GSH. Before use, the enzyme was activated by incubating in 200 μM DTT on ice for 15 min. A further 200-fold dilution in caspase-1 buffer [100 mM Hepes, pH 7.5/10% (w/v) glycerol/0.1% (w/v) CHAPS/0.1 mM EDTA] was made to overcome the reduction power of DTT which resulted in a final caspase-1 concentration of approximately 4 nM. The final reaction contained 14 μM Ac-YVAD-AMC substrate and various concentrations of DSF and/or DTT as indicated in Figure 3A, in a total volume of 100 μL. Proteolytic activity was monitored in a Fluoroskan II plate reader at 37 °C as previously described.
Purification of caspase-3 was performed as described earlier. The purified protein was stored until use in 100 mM Hepes, pH 7.5/10% (w/v) sucrose/0.1% (w/v) CHAPS/0.1 mM EDTA/10 mM DTT at −70 °C. To eliminate DTT from the caspase-3 storage buffer, the enzyme was diluted 10-fold in the same buffer (minus DTT) and dialyzed for 3 h in a nitrogen atmosphere, using Millipore V membranes (0.025 μm). The enzyme was subsequently diluted another 20-fold in the storage buffer (minus DTT) to a final concentration of approximately 0.2 nM. Final concentrations of DTT were 1−2 μM as determined by monobromobimane conjugation and HPLC separation. Enzymatic activity was assayed with 40 μM Ac-DEVD-AMC and various concentrations of DSF and DTT in a total volume of 100 μL with a Fluoroskan II plate reader at 37 °C as described above

SDS−PAGE and Western Blotting. [1]
Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) was performed using Biorad's minigel system as instructed. The gel (12% polyacrylamide) was run under nonreducing conditions (without β-mercaptoethanol). Protein samples were then transferred to a nitrocellulose membrane using a conventional electrophoretic Western blotting technique. The membrane was dried, and an X-ray film was exposed for 8−9 days before developing.

[1]. Disulfiram is a potent inhibitor of proteases of the caspase family. Chem Res Toxicol. 1997 Dec;10(12):1319-24.

[2]. Caspases mediate 6-hydroxydopamine-induced apoptosis but not necrosis in PC12 cells. J Neurochem. 1998 Jun;70(6):2637-40.

[3]. Yakovlev AA, Gorokhovatsky AY, Onufriev MV, Beletsky IP, Gulyaeva NV. Brain cathepsin B cleaves a caspase substrate. Biochemistry (Mosc). 2008 Mar;73(3):332-6.

We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide. [1]

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The neurotoxin 6-hydroxydopamine (6-OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6-OHDA-induced apoptosis is morphologically indistinguishable from serum deprivation-induced apoptosis. Exposure of PC12 cells to a low concentration of 6-OHDA (25 microM) results in apoptosis, whereas an increased concentration (50 microM) results in a mixture of apoptosis and necrosis. We investigated the involvement of caspases in the apoptotic death of PC12 cells induced by 6-OHDA, using a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), and compared this with serum deprivation-induced apoptosis, which is known to involve caspases. We show that zVAD-fmk (100 microM) completely prevented the apoptotic morphology of chromatin condensation induced by exposure to either 6-OHDA (25 and 50 microM) or serum deprivation. Furthermore, cell lysates from 6-OHDA-treated cultures showed cleavage of a fluorogenic substrate for caspase-3-like proteases (caspase-2, 3, and 7), acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin, and this was inhibited by zVAD-fmk. However, although zVAD-fmk restored total cell viability to serum-deprived cells or cells exposed to 25 microM 6-OHDA, the inhibitor did not restore viability to cells exposed to 50 microM 6-OHDA. These data show the involvement of a caspase-3-like protease in 6-OHDA-induced apoptosis and that caspase inhibition is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6-OHDA neurotoxicity. [2]

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We show that an enzyme exists in rat brain capable of cleaving the caspase-3 specific peptide substrate Ac-DEVD-AMC at low pH. The enzyme shows properties of a cysteine protease and is localized, predominantly, in lysosomes. We have purified this enzyme from rat brain and identified it by MALDI-TOF MS. The enzyme possessing "acidic" DEVDase activity in rat brain appears to be cathepsin B. It remains obscure, whether cathepsin B participates in cleavage of caspase-3 substrates in vivo. We suggest that under certain conditions (e.g. in hypoxia) cathepsin B participates in cleavage of caspase-3 substrates in brain cells. [3]

C30H37N5O13

675.64048

675.238

169332-61-0

9896164

White to off-white solid powder

1.4±0.1 g/cm3

1200.1±65.0 °C at 760 mmHg

679.6±34.3 °C

0.0±0.3 mmHg at 25°C

1.594

1.07

8

13

17

48

1330

4

CC1=CC(=O)OC2=C1C=CC(=C2)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)C

ALZSTTDFHZHSCA-RNVDEAKXSA-N

InChI=1S/C30H37N5O13/c1-13(2)26(35-27(44)18(7-8-22(37)38)33-29(46)19(11-23(39)40)31-15(4)36)30(47)34-20(12-24(41)42)28(45)32-16-5-6-17-14(3)9-25(43)48-21(17)10-16/h5-6,9-10,13,18-20,26H,7-8,11-12H2,1-4H3,(H,31,36)(H,32,45)(H,33,46)(H,34,47)(H,35,44)(H,37,38)(H,39,40)(H,41,42)/t18-,19-,20-,26-/m0/s1

(4S)-4-[[(2S)-2-acetamido-3-carboxypropanoyl]amino]-5-[[(2S)-1-[[(2S)-3-carboxy-1-[(4-methyl-2-oxochromen-7-yl)amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid

Ac-DEVD-AMC; 169332-61-0; Ac-Asp-Glu-Val-Asp-AMC ammonium salt; CPP32/Apopain Substrate; Ac-Asp-Glu-Val-Asp-AMC; N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin; (4S)-4-[[(2S)-2-acetamido-3-carboxypropanoyl]amino]-5-[[(2S)-1-[[(2S)-3-carboxy-1-[(4-methyl-2-oxochromen-7-yl)amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid; Ac-DEVD-AMC ammonium salt;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~0.67 mg/mL (~0.99 mM)

Solubility in Formulation 1: 3.33 mg/mL (4.93 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)