| Autor: |
Nobel, C. S. I., Kimland, M., Nicholson, D. W., Orrenius, S., Slater, A. F. G. |
| Zdroj: |
Chemical Research in Toxicology; December 16, 1997, Vol. 10 Issue: 12 p1319-1324, 6p |
| Abstrakt: |
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 of the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts prepared from CD95-activated Jurkat cells. Since reduced diethyldithiocarbamate had no effect and DSF 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 (Kapp) for this inhibition was estimated to be 0.45 × 103 M-1 s-1. DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1β-converting enzyme, ICE), with a Kapp of 2.2 × 103 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. |
| Databáze: |
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