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Metallothioneins (MTs) release bound metals when exposed to nitric oxide. At inflammatory sites, both metallothionein and inducible nitric oxide synthase (iNOS) are induced by the same factors and the zinc released from metallothionein by NO suppresses both the induction and activity of iNOS. In a search for a possible modulatory mechanism of this coexpression of counteracting proteins, we investigated the role of the glutathione redox state in vitro because the oxidation state of thiols is involved in the metal binding in Cd-S or Zn-S clusters found in metallothioneins, and NO also binds to reduced glutathione via S-nitrosation. Using a variety of techniques, we found that NO and also ONOO --mediated metal release from purified MTs is suppressed by reduced glutathione (GSH), but not by oxidized glutathione. Considering the millimolar concentrations of GSH present in mammalian cells, the metal release from MTs by NO should play no role in living systems. Therefore, the fact that it has been observed in vivo points to a hitherto unknown mechanism or additional compound(s) being involved in this physiologically relevant reaction and as long as this additional factor is not found experimental results on the MT-NO interaction should be treated with caution. Contrary to the peroxynitrite-induced activation of guanylyl cyclase, where GSH is needed, we found that the metal release from metallothionein by peroxynitrite is not enhanced, but also suppressed by reduced glutathione. In addition, we show that zinc, the major natural metal ligand in mammalian MTs and suppressor of iNOS, is released more readily under the influence of NO than cadmium, but in contrast to the MT isoform 1, the amount of metal released from the b-domain of MT-2 is comparable to that from the a-domain.Keywords: glutathione; metallothionein; nitric oxide; NMR spectroscopy; SEC-ICPMS.Metallothioneins (MTs) are a family of small (6-7 kDa) metal-binding proteins [1][2][3] with the highest known metal content after ferritins. The high amount of cysteine residues in MTs (30% of all amino acids are cysteine) allows these proteins to coordinate multiple mono (Cu + , Ag + ) or divalent metals (Zn 2+ , Cd 2+ ). Mammalian MTs bind seven divalent metals in two separate domains [4]. Three metals are bound in an M 3 Cys 9 cluster in the N-terminal b-domain, while an M 4 Cys 11 four metal cluster is formed in the C-terminal a-domain [4]. Of the four known mammalian MT isoforms [2], the two best studied and most widely occurring isoforms (1 and 2) are most abundant in parenchymatous tissues, i.e. liver, kidney, pancreas and intestines [5][6][7] . The naturally bound metal zinc can be displaced by cadmium up to about 5 mol per mol protein by simple addition of Cd 2+ [13] in vitro. Living animals fed a cadmium-rich diet produce a mixed-metal MT with zinc bound preferentially in the b-and cadmium in the a-domain [11,13,14]. The artificial Cd 7 -MT can only be obtained after complete zinc removal by lowering the pH [15] in vitro.Although the biological function(s) o...

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