Modulation of nitric oxide-mediated metal release from metallothionein by the redox state of glutathione in vitro Leila Khatai 1 , Walter Goessler 2 , Helena Lorencova 2 and Klaus Zangger 1 1 Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Austria; 2 Institute of Chemistry, Analytical Chemistry, University of Graz, Austria 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 oxi- dation 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 addi- tional 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 [1–3] 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 [5–7] but their occurrence and biosynthesis have been documented in many tissues and cell types. The 3D structures of MT1 [8] and MT2 [9–12] are very similar, but there are various indications of increased flexibility and metal mobility in the b-domaininMT-1[8].Thenaturally 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) of MTs still remain somewhat elusive [16], they have been proposed to play an important role in zinc homeostasis [1,17] and heavy metal detoxification [18,19], although the latter is probably not an evolutionary conserved function but rather a property of these cysteine-rich proteins. Due to the different metal affinities for zinc and cadmium in the two separate domains [13], the b-domain has been implicated in zinc homeostasis and the tight binding of cadmium in the a-domain was proposed to be responsible for the role of MTs in heavy metal detoxification. In addition, it has been reported that MTs act as radical scavengers under oxidative stress [20–22]. Another possible key player in the role of MTs in signal transduction might be nitric oxide (NO), which was shown recently, both in vitro [23–25] and in vivo [26–29], to interact with MTs and thereby releases bound zinc and cadmium. The importance of MTs in NO-induced changes in intra- cellular zinc homeostasis has been reported by St Croix et al.[30]. Correspondence to K. Zangger, Institute of Chemistry/Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria. Fax: + 43 316 380 9840, Tel.: + 43 316 380 8673, E-mail: klaus.zangger@uni-graz.at Abbreviations: DEA/NO, 2-(N,N-diethylamino)-diazenolate-2-oxide- Na; GSH, reduced glutathione; GSSG, oxidized glutathione; iNOS, inducible NO-synthase; MT, metallothionein; NO, nitric oxide; SEC–ICPMS, size exclusion chromatography–inductively coupled plasma mass spectrometry; SIN-HCl, 3-morpholinosydnoni- mine.HCl. (Received 26 February 2004, revised 6 April 2004, accepted 14 April 2004) Eur. J. Biochem. 271, 2408–2416 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04160.x Based on the preferred release of metal from the b-domain of mouse MT1, where zinc is preferentially bound in vivo, we suggested recently that MTs had anti-inflamma- tory activity [31]. This activity relies on the suppression of the expression and activity of inducible nitric oxide synthase (iNOS) by zinc [32,33], released from MT under the influence of nitric oxide (NO), and the scavenging of NO through covalent binding to MTs [23] to form S-nitroso- thiols. Such a role of MTs in the inflammatory response has been corroborated by the significantly altered inflammatory behavior during experimental autoimmune encephalomye- litis [34] observed in MT deficient mice. In addition, it has been reported that overexpression of MT reduces the sensitivity of eukaryotic cells to oxidative injury [35] and the cytotoxic effects of NO [29]. As both iNOS and MTs are induced at inflammatory sites by the same compounds and MT scavenges NO and suppresses its production, one starts to wonder why they are both produced at inflammatory sites but counteract each other. Therefore, we looked for a possible regulatory mechanism for the interplay between NO production and metal release from MTs in order to understand this dual expression of opposing proteins. As the metal is held in place by thiolate ligands [4] in MTs, other thiols may well influence the metal-bond formation and breakage. The major low-molecular mass thiol com- pound in plants and animals is the tripeptide L -c-glutamyl- L -cysteinyl-glycine also known as glutathione (GSH in reduced form and GSSG in its oxidized form). Glutathione has also been described as a Ôtransport peptideÕ in vivo for NO through the formation of S-NO groups [36]. The glutathione redox couple, a cellular redox buffer which maintains the given thiol/disulfide redox potential, has already been implicated in modulating the metal release from metallothionein in the absence of nitric oxide by Vallee, Maret and coworkers [22,37,38]. These authors reported increased metal release in the presence of oxidized glutathione (GSSG) and even slightly tighter metal binding under the influence of reduced glutathione (GSH) [37]. We investigated in vitro the effect of GSH/GSSG on NO- mediated metal release of MT2 by circular dichroism (CD) spectroscopy, size exclusion chromatography–inductively coupled plasma mass spectrometry (SEC–ICPMS) and nuclear magnetic resonance (NMR) spectroscopy. In a previous study [31], we used Cd 7 -MT1 to study the structural consequences of NO exposure on MTs by 1 H and 113 Cd-NMR spectroscopy as Zn cannot be studied by regular NMR experiments. However, Cd 7 -MT1 is never found in natural sources and differences in metal-binding constants between Cd and Zn might prevent the inter- pretation of in vivo processes with data obtained on an artificially cadmium-enriched protein. Therefore, we have limited the present study to Zn 7 -MT2 and Cd 5 Zn 2 -MT2, which have been isolated from natural sources. In addition to nitric oxide, peroxynitrite (ONOO – )may also play a significant role in the metal release from MTs, as it has been suggested that the decomposition of peroxy- nitrite at physiological pH constitutes the actual component of NO cytotoxicity [39]. A widespread signal transduction mechanism for NO involved in, e.g. platelet aggregation, blood pressure control and neurotransmission functions via stimulation of guanylyl cyclase [40]. In contrast to NO, glutathione-dependent bioactivation of peroxynitrite is involved in enzyme stimulation and this points again at a possible key role of glutathione in the NO and/or ONOO – mediated metal release from MTs. Materials and methods The NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide- Na (DEA/NO) and the peroxynitrite donor 3-morphol- inosydnonimine.HCl (SIN-HCl) were purchased from Alexis Biochemicals (Lausen, Switzerland). Due to the limited lifetime of DEA/NO at low to moderate pH, stock solutions were prepared in 10 m M NaOH (pH 12). By adding such stock solutions to neutrally buffered systems, 1.5 mol equivalents of NO are released from DEA/NO with a half-life of 16 min at 24 °C. Using molecular oxygen, SIN-HCl generates superoxide and NO, which spontaneously combine to form peroxynitrite. A fresh solution of peroxynitrite was prepared according to a published procedure [41] and stored at )80 °C until used. Rabbit liver metallothionein-2 in the zinc form (Zn 7 -MT2) and a mixed cadmium, zinc form (Cd 5 Zn 2 -MT2) were obtained from Sigma (Vienna, Austria). We reconstituted Zn 7 -MT2 to ascertain the stochiometry of seven metals per protein monomer according to the procedure des- cribed by Vas ˇ ak [15], but found no differences in its behavior to the unpurified commercially available product. All other chemicals were purchased from Sigma (Vienna, Austria) at the highest purity available. Due to problems associated with the use of organic buffers in inductively coupled plasma mass spectrometry (ICPMS) instruments and protonated buffers for NMR, we used aqueous phosphate buffers for all experiments (see below). To evaluate a possible influence of phosphate ions on MT2 during these experiments, the CD experiments were also performed in 20 m M Hepes buffer, but showed the same results. CD spectroscopy The complete absence of aromatic amino acids in metallothioneins allows the use of UV and CD spectros- copy to observe the cadmium-thiolate charge transfer transition, which occurs around 250 nm [42]. This region is usually completely masked by aromatic groups. CD spectra were recorded on a Jasco J-715 spectropolarimeter andanalyzedusingtheprogram CDSCAN . For each wavelength scan, the average was taken from 10 accumu- lations with the following parameters: step resolution, 0.2 nm; speed, 50 nmÆmin )1 ; response time, 1 s; band- width, 2 nm. For time scans, we used: wavelength, 260 nm; step resolution, 1 s; response time, 1 s; band- width, 2 s. Samples consisted of 100 l M Cd 5 Zn 2 -MT2 in 20 m M potassium phosphate buffer at pH 7.5. Stock solutions of GSH (50 m M in phosphate buffer, pH 7.5), GSSG (50 m M in phosphate buffer, pH 7.5) and DEA/ NO (20 m M in 10 m M NaOH) were added to the MT2 solution to give final concentrations of 1 m M of each compound in the respective spectra. The range between 230 and 300 nm was recorded for the CD spectra and time scans were obtained by monitoring the CD at the maximum of the cadmium-thiolate charge transfer band at 260 nm for 20 min after mixing the components. Ó FEBS 2004 NO-mediated metal release from metallothionein (Eur. J. Biochem. 271) 2409 SEC–ICPMS ICPMS enables the determination of a variety of elements in solution. In order to differentiate between protein-bound and free metal, a preceding separation of protein and unbound metal by size exclusion chromatography (SEC) is necessary. Instead of performing these two steps separately, thecouplingofSECandICPMSoffersaveryelegant alternative [43,44]. For our studies, a Pharmacia Superdex 75 PC 3.2/30 gel filtration column was connected to an Agilent HP 1100 ChemStation SEC system (Agilent, Waldbronn, Germany) equipped with a UV monitor set to 220 nm. The outlet of the UV-detector was connected directly via a PEEK capillary (i.d. 0.12 mm, length 90 cm) to the l-flow PFA-100 nebulizer (CPI International, Santa Rosa, USA) of the Agilent 7500c ICPMS system. The isotopes 64,66 Zn and 111,114 Cd were monitored. All meas- urements were performed at least twice and the averages were taken over both isotopes of zinc and cadmium, respectively. A 20 m M aqueous ammonium phosphate buffer, pH 6.5 was used as eluent at a flow rate of 0.1 mLÆmin )1 . MT2, GSH and GSSG solutions were made metal-free by washing through a Chelex-100 column (Sigma, Vienna, Austria) and stored in polyethylene flasks. Samples of 1 lL were injected onto the column and separated at 22 °C. All solutions were filtered and degassed by N 2 bubbling prior to use. A solution of 20 l M Cd 5 Zn 2 - MT2 was mixed with stock solutions of 2 m M DEA/NO, 2m M SIN-HCl, 2 m M ONOO – ,10m M GSH or 5 m M GSSG and then equilibrated for at least 15 min prior to injection onto the gel filtration column. NMR spectroscopy Series of two-dimensional TOCSY [45] NMR spectra were recorded on a Varian Unity INOVA 600 MHz NMR spectrometer at 25 °C. The water signal was suppressed with the WATERGATE sequence [46]. For each of the 256 increments, 2048 complex data points were recorded. The data were multiplied with a 60° phase-shifted, squared sine- bell window function in both dimensions prior to Fourier transformation. The total experimental time of one 2D spectrum was 12 h. Samples consisted of 2.5 mg of Zn 7 - MT2 or Cd 5 Zn 2 -MT2 in 0.5mL of 20m M potassium phosphate buffer pH 6.5 and 50 lLD 2 O. A stock solution of 50 m M DEA/NO was added directly to the NMR samples to give final concentrations of 0.2, 0.5, 1 and 3 m M DEA/NO. After each addition, the solution was equili- brated for at least 20 min prior to the start of the NMR acquisition. The same experiment was performed in the presence of GSH, whereby the GSH concentration was 1m M for samples containing 0.2 and 0.5 m M DEA/NO and 5m M GSH was added when the DEA/NO concentration was 1 and 3 m M . Results CD spectroscopy The Cd-S charge transfer transitions are responsible for the absorption and CD above 230 nm in UV and CD spectra of metallothioneins devoid of any aromatic residues that usually obscure this spectral region. By monitoring this charge transfer band, CD spectroscopy was first used by Ka ¨ gi and coworkers to follow the metal-binding stochiom- etry of MTs [42]. To study the metal release by NO and its modulation by GSH and GSSG, a solution of 100 l M Cd 5 Zn 2 -MT2 was exposed to NO for 20 min by adding DEA/NO at a final concentration of 1 m M . For reduced and oxidized glutathione, concentrations of 1 m M were used. CD spectra of the range between 230 and 300 nm are shown in Fig. 1A. The Cd-S charge transfer band at 260 nm is reduced clearly after the addition of NO, indicating the breaking of cadmium-cysteine bonds and therefore release of cadmium. The presence of GSH reduces the metal release almost completely, while GSSG even slightly increased the cadmium release by nitric oxide. The decay occurs in the first 10 min after the addition of NO as observed by monitoring the CD at the maximum of the charge transfer band at 260 nm (Fig. 1B). The lower molar ellipticity at time 0 in the GSSG/NO treated sample derives from partial metal release during the period from mixing the solutions until the start of the data acquisition. While with the CD measurements nothing can be said about the faith of zinc bound in Cd 5 Zn 2 -MT2 after the addition of NO, the amount of cadmium is reduced at this rather extreme NO Fig. 1. Wavelength and time scans of the CD of MT2 in the presence and absence of NO and GSH/GSSG. CD spectra of 100 l M Cd 5 Zn 2 - MT2 alone or with 1 m M GSH in 20 m M potassium phosphate buffer, pH 7.5 upon the exposure to 1 m M DEA/NO are shown in (A). The molar ellipticity ([h]inkdegÆdmol )1 Æcm )2 )atthemaximumoftheCd-S charge-transfer band at 260 nm as a function of time can be seen in (B). 2410 L. Khatai et al.(Eur. J. Biochem. 271) Ó FEBS 2004 concentration by at most 2 cadmium atoms per MT2 molecule. SEC–ICPMS CD spectroscopy enables the monitoring of breaking Cd-S bonds, but it does not give information about released metals, because there might be a situation when some metal- thiolate bonds are broken, but the metal is still held in place by remaining Cd-S bonds. In addition, no information about bound zinc in the mixed metal MT2 is obtained. Both uncertainties can be clarified with SEC–ICPMS [43,44]. The sample is applied to a gel filtration column, which separates free from protein-bound metal and subsequently both zinc and cadmium levels are determined by ICPMS. Stock solutions of 2 m M DEA/NO, 10 m M GSH and 5 m M GSSG were added to samples of 20 l M Cd 5 Zn 2 -MT2 to give final ratios as indicated at the bottom of Fig. 2. The normalized amounts of zinc and cadmium in the MT2 fraction, taking into consideration the dilution effects by adding stock solutions of GSH, GSSG and DEA/NO (Fig. 2) clearly show that the release of both cadmium and zinc by NO is suppressed completely by GSH, but not GSSG. As already suggested in our previous paper [31], zinc is more readily released than cadmium. Rather high concentrations of NO are needed to observe significantly reduced cadmium levels in MT2, which corroborates the role of MTs in heavy metal detoxification as a result of rather tight binding of cadmium to MTs [18,19]. The maximum number of metals released from Cd 5 Zn 2 -MT2 at the highest NO concentrations used in these ICPMS studies amount to 1.5 Zn and 3.25 Cd per MT2 molecule, which shows that in contrast to mouse MT1, [31] significant amounts of metal are also set free from the a-domain. The clean separation of MT2 from GSH, GSSG and DEA/NO can be seen in representative UV traces recorded after the gel-filtration step. Dilution effects are partly responsible for slight differences in both these UV traces as well as 66 Zn and 114 Cd intensities between the pure MT2 sample and mixtures with DEA/NO and GSH (Fig. 3). The disappearance of small amounts of MT2 dimers by adding NO may be a result of disrupting S-Cd-S bonds in these presumably metal-bridged dimers [47,48]. The zinc and cadmium released from MT2 are not seen in these chromatograms due to slight binding to the column. However, they could be detected as very broad peaks in subsequent runs or be removed from the column with weak metal chelators, like cysteine (not shown). A major molecule of NO toxicity under physiological conditions is ONOO – whose function in the stimulation of guanylyl cyclase requires the presence of reduced gluta- thione [39]. To elucidate the possible role of peroxynitrite in the metal release from MTs in the presence of reduced and oxidized glutathione we carried out SEC–ICPMS measure- ments on a series of solutions containing a mixture of Cd 5 Zn 2 -MT2 (20 l M stock solution), the peroxynitrite donor SIN-HCl (2 m M stock solution), a freshly prepared peroxynitrite solution (2 m M stock solution) and either GSH (10 m M stock solution) or GSSG (10 m M stock solution) at the ratios shown in Fig. 4. As can be seen, the metal release by both SIN-HCl and ONOO – is not as pronounced as for NO itself, leading to a maximum of about 1.2 Zn at far from physiological NO/MT ratios of 100 : 1 and only insignificant amounts of cadmium being released at the highest ONOO – concentration. Even more interestingly, in contrast to the peroxynitrite-mediated activation of the guanylyl cyclase the presence of GSH does not lead to enhanced but lower levels of metal release, thus pointing to a fundamentally different mode of action. NMR spectroscopy To obtain domain-specific structural information about the metal release from MTs and its regulation by GSH series of 2D TOCSY spectra [45] were acquired. Thereby, we titrated a solution containing 0.6 m M rabbit Cd 5 Zn 2 -MT-2, rabbit Zn 7 -MT-2 or rabbit Zn 7 -MT-2 + GSH with different concentrations of DEA/NO 0, 0.2, 0.5, 1, 3 m M . After each addition of DEA/NO, the solution was equilibrated for at least 20 min and subsequently the 2D spectrum recorded during 12 h resulting in a total experimental time of 2.5 days for one full titration. In the 2D TOCSY spectra, Fig. 2. Histograms showing the normalized Zn and Cd contents in the protein fraction of the SEC–ICPMS chromatograms with relative error bars in the presence of NO, GSH and/or GSSG. A solution of 20 l M Cd 5 Zn 2 -MT2 was diluted with stock solution of 2 m M DEA/NO, 10 m M GSH and 5 m M GSSG to give ratios of these compounds as indicated at the bottom. Ó FEBS 2004 NO-mediated metal release from metallothionein (Eur. J. Biochem. 271) 2411 only well-resolved peaks were integrated and their signal intensities normalized to the intensity in the absence of NO (I 0 ). Representative NO-concentration dependences for all well-resolved signals from the a- (22 peaks) and b-domain (31 peaks) were averaged and are shown in Fig. 5. The reductions in proton signal intensities reflect the increase of dynamic processes when metal is released and/or the conformational variety in the disulfide bridged MT2 formed after NO treatment as described [31] and so it can be used indirectly to follow metal binding stochiometries. The addition of NO at these high concentrations leads to signal reductions both in the a-andtheb-domains of Zn 7 -MT2 and Cd 5 Zn 2 -MT2 with however, larger decays in Zn 7 -MT2. As expected, based on the observations from CD-spectros- copy and SEC–ICPMS measurements, GSH led to a significant reduction in signal losses. A more quantitative estimate of proton signal reductions as a function of time can be obtained from the signal reductions in a well-resolved signal that are shown for the two domains separately in Fig. 6. In contrast to mouse Cd 7 -MT1 [31], there is a reduction in signal intensities of a similar magnitude from both a-andb-domains with NO. Obviously the differences in metal binding strength between the two separate domains is more pronounced in MT1 than MT2. This is corrobor- ated by the higher flexibility observed in the b-domain of MT1 [8], based on increased NH and cadmium-cadmium exchange rates and the low number of NOEs observed in the b-domain of mouse Cd 7 -MT1 compared with the a-domain. Recently, Maret and coworkers found a large difference in the amount of metal released by NO in the two domains of MT3 [49]. Zinc from the b-domain was set free much easier than from the a-domain. Thus, the already observed distinctive metal mobilities in b-domains of MT isoforms 1, 2 and 3, which follow the order MT3 > MT1 > MT2 [8,50,51] are mirrored in the metal release upon NO exposure. Discussion The presented results show clearly that the metal release from MT2 by nitric oxide and peroxynitrite is suppressed by reduced but not oxidized glutathione. Due to different requirements of sample concentrations in the presented experiments, the interaction of MT2, NO, ONOO – and GSH has been established for ratios ranging from 1:0.3:1.4upto1:10:100(MT:NO/ONOO – :GSH) with MT2 being between 20 and 600 l M . The reason for NO protection by glutathione could be attributed to its faster reaction with NO or the reported binding of GSH in the b-domain of metallothionein [52,53] and thus the blocking of certain nitrosation sites. Surprisingly, we did not observe any changes in the TOCSY NMR spectra upon the addition of GSH (data not shown), which is indicative of no specific binding under the conditions (buffer system and pH) used here. Still, the suppression of the NO–MT2 interaction by GSH may be a result of faster reaction with glutathione [54], the binding of GSH to MT2 or a combination of both. GSNO which is formed in the Fig. 3. UV traces and online element-selective detection of the SEC–ICPMS characterization of pure 20 l M Cd 5 Zn 2 -MT2 and mixtures of MT2 + NO (1 +11) and MT2 + GSH + NO (1 +25 +10). The amount of zinc and cadmium is shown as a function of retention time and is given as counts per second with the higher number for cadmium representing its higher sensitivity on the ICPMS system used. The extinction in the UV trace is given in mAU. 2412 L. Khatai et al.(Eur. J. Biochem. 271) Ó FEBS 2004 reaction of NO with GSH serves as a carrier for NO in vivo and acts as an NO-donor that undergoes spontaneous homolytic release of NO radicals [36,55,56]. Under physiological conditions, concentrations of NO between 0.1 and 4 l M have been described [54]. Considering that the GSH concentration in mammalian cells varies over the range of 0.5–10 m M [57] our results suggest that the NO and ONOO – -mediated metal release from metallothionein should play no significant role in living systems. The amount of NO and oxidized glutathione increases during inflammation [58,59] but we are not aware of any report of GSH concentrations low enough to enable metal release from MT2 upon the exposure to nitric oxide or peroxy- nitrite. However, a number of reports have been published demonstrating the physiological significance of the NO–MT interaction and in particular the metal release in vivo.Using a fluorescent MT2 fusion protein, a conformational change in MT2, indicative of metal- release, has been observed by Pearce et al. [28] after the administration of NO or NO- stimulating factors in endothelial cells. The metal release itself has been studied in cultured epithelial cells [26]. Metallothionein has also been shown to protect eukaryotic cells from the cytotoxic and DNA-damaging effects of nitric oxide [29]. So, while the binding of NO to GSH in vivo does not obviously prevent NO from interacting with MT2, we have shown that in vitro it suppresses the metal release from metallothioneins. This points to an hitherto unknown mechanism or compound(s) being involved in this inter- action in living cells and information about this additional factor is needed in order to perform physiologically relevant future in vitro studies and in the interpretation of results obtained from in vivo experiments on the NO–MT interaction. As predicted earlier [31], zinc is more readily released from MTs than cadmium, which is probably a combination of tighter binding of cadmium than zinc in metallothioneins and the preference of zinc in the more flexible b-domain. In addition to the already described differences in flexibility of the b-domain in MT isoforms 1 and 2 [8], we found that the domain specific distinctions upon NO exposure are less Fig. 4. Dilution-corrected Zn and Cd contents in the MT2 fraction. Histograms of normalized, dilution-corrected Zn and Cd contents in the MT2 fraction of SEC–ICPMS chromatograms obtained by applying mixtures of stock solutions of 20 l M Cd 5 Zn 2 -MT2, 2 m M SIN-HCl, 2 m M ONOO – ,10m M GSH and 5 m M GSSG yielding final ratios as indicated. Fig. 5. Histograms of NMR proton peak intensity changes of 0.6 m M solutions of either Zn 7 -MT2 (+GSH) or Cd 5 Zn 2 -MT2 b-(top) and a-domain (bottom) exposed to NO in the absence or presence of 5 m M GSH. The average intensities of all intense, well-resolved peaks in the 2D TOCSY spectra (22 peaks from the b-domain and 31 from the a-domain) with 0 and 3 m M DEA/NO were used. Ó FEBS 2004 NO-mediated metal release from metallothionein (Eur. J. Biochem. 271) 2413 significant in MT2 unlike previously found for mouse Cd 7 - MT1 [31]. In conclusion, we have shown that reduced but not oxidized glutathione suppresses the NO and ONOO – - mediated metal release from metallothionein in vitro and that zinc is indeed more readily released under these conditions as suggested earlier [31]. The millimolar concen- trations of GSH present in mammalian cells should thus eliminate any nitric oxide or peroxynitrite mediated metal release from MTs. However, as such an interaction has been found in vivo, an unknown mechanism or compound must also be involved in this interaction. Therefore, we believe that results from both in vivo and in vitro studies on the NO–MT interaction should be interpreted with caution for as long as this discrepancy has not been resolved. Acknowledgements This work has been supported by the Austrian Science Foundation (Project No. P15289 to K. Z.). We would like to thank Monika Oberer for help in recording the CD spectra and Regina Golser for helpful discussions. References 1. Ka ¨ gi,J.H.&Scha ¨ ffer, A. 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