Modulation of nitric oxide-mediated metal release from metallothionein by the redox state of glutathione in vitro.
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 beta-domain of MT-2 is comparable to that from the alpha-domain.
Khatai
Leila
L
Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Austria.
Goessler
Walter
W
Lorencova
Helena
H
Zangger
Klaus
K
D000818:Animals; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013455:Sulfur / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D020030:Nitric Oxide Donors; D020033:Protein Isoforms; D002104:Cadmium; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D013455:Sulfur; D008668:Metallothionein; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D005978:Glutathione; D015032:Zinc