Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liver

Palumaa, Peep; Mackay, Elaine A.; Vašák, Milan

doi:10.1021/bi00122a040

Cited by 39 publications

(30 citation statements)

References 32 publications

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“…During the purification, the sea urchin MT exhibited dimerisation which we believe to be similar in nature to the dimerisation reported by Palumaa et al (1992) as the addition of EDTA to the larger-molecular-mass material resulted in a reduction of the apparent molecular mass to that of the lower-molecular-mass peak. It may be that in this case phosphate ions are also involved in this dimerisation phenomena as when E. coli cells were sonicated in phosphate buffer the ratio of dimer to monomer was approximately 1 : 1 whereas when Tris/HCl buffer was used the ratio of dimer to monomer was greatly reduced (Fig.…”

Section: Discussionsupporting

confidence: 81%

“…Two major cadmium peaks were obtained having apparent molecular masses of approximately 8 kDa and 15.5 kDa. Incubation of the larger molecular mass material (15.5 kDa) with 1 mM EDTA, pH 8.0, resulted in a reduction of the molecular mass to a value equal to the smaller peak (8 kDa) suggesting that these peaks represented a monomer and a metal-mediated dimer, as shown previously for rabbit liver MT (Palumaa et al, 1992). The cadmium-containing fractions were pooled, as indicated in Fig.…”

Section: Expression and Purification Of The Recombinant Sea Urchin Mtsupporting

confidence: 53%

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Purification and Characterisation of Recombinant Sea Urchin Metallothionein Expressed in Escherichia Coli

Wang¹,

Mackay²,

Kurasaki³

et al. 1994

European Journal of Biochemistry

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Metallothioneins (MT) are metalloproteins expressed tissue specifically during the development of the sea urchin, Strongylocentrotus pururutus. To explore their structural and functional features and to compare them with those of the evolutionary distant mammalian MTs, one isoform (MTA) was obtained as the cadmium-containing form, from synthetic cDNA heterologously expressed in Escherichia coli. The purified protein was identified as the desired product by a combination of peptide-map analysis, amino acid sequence analysis and ion-spray mass spectroscopy. The existence of seven " T d NMR resonances revealed that the recombinant protein binds seven Cd ions/molecule. The position of the NMR resonances (605 -695 ppm) and the electronic absorption features suggest that the sea urchin MTA, like the mammalian MTs, possesses tetrahedrally coordinated cadmiumthiolate clusters. With its large Stokes' radius, sea urchin MTA resembles the mammalian forms, suggesting a comparable elongated molecular shape. Measurements by spectrophotometric pH titration of cadmium binding by the recombinant protein suggest that it possesses two metal-thiolate clusters of distinctly different stability. At pH 7 the average apparent association constant for Cd2' in the clusters is about 20-times weaker in sea urchin MTA than in rabbit MT-2.Metallothioneins (MT) are widely found low-molecularmass, cysteine-rich proteins distinguished by an exceptionally high content of d' O metal ions (Zn", Cd2+ and Cu'+) (see Roesijadi, 1993, andKagi, 1993, for reviews). MTs have traditionally been thought to be involved in cellular detoxification of metals but more central functions such as an involvement in the regulation of Zn-dependent processes in gene expression (Zeng et al., 1991a and and in development (Nemer et al. 1984;De et al., 1991) have also been suggested.The sea urchin Strongylocentotus pupurutus like many other species, contains a family of MT genes. Two of these genes, designated MTA and MTB,, have been well characterised. These genes show a high degree of identity, but probes specific for their mRNAs have been constructed and used to show that the expression patterns of the genes are quite distinct. Both mRNAs begin to accumulate at the early blastula stage (12 h after fertilization) but the MTB, mRNA becomes localized in the embryonic gut and in the oral ectoderm while the MTA mRNA is spatially restricted to the aboral ectoderm (Nemer et al., 1991). As found for mammalian MTs, both forms are readily inducible by zinc although with unequal thresholds (Wilkinson and Nemer, 1987). This responsiveness to zinc supply is accounted for by the occurrence of at least six metal-regulatory elements (MRE) in the 5' upstream region of both the sea urchin genes (Bai et al., 1993). The two MREs of the MTA gene (designated a and b) nearest to the transcription start site are identical in both sequence and position (relative to the TATA box) to those which give maximal activity in mammalian systems (Harlow et al., 1989). In addition to this conservation of...

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Section: Discussionsupporting

confidence: 81%

Section: Expression and Purification Of The Recombinant Sea Urchin Mtsupporting

confidence: 53%

Purification and Characterisation of Recombinant Sea Urchin Metallothionein Expressed in Escherichia Coli

Wang¹,

Mackay²,

Kurasaki³

et al. 1994

European Journal of Biochemistry

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“…Such a selfassociation with an association constant of 3 ϫ 10 4 M Ϫ1 at pH 6.8 (11) is consistent with the dimerization of MT observed in the crystal structure (4) and with hydrodynamic properties of both Zn-MT and Cd-MT (11,34). The relatively fast rate of zinc exchange between MT and Gal4 suggests that this process is also controlled by contact between the two molecules and ligand swapping.…”

Section: Resultssupporting

confidence: 72%

Coordination dynamics of biological zinc “clusters” in metallothioneins and in the DNA-binding domain of the transcription factor Gal4

Maret

Larsen

Vallée

1997

Proc. Natl. Acad. Sci. U.S.A.

110

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The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (k fast Ӎ 5000 min ؊1 ⅐M ؊1 ; k slow Ӎ 200 min ؊1 ⅐M ؊1 , pH 8.6, 25؇C) that are thought to ref lect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k Ӎ 800 min ؊1 ⅐M ؊1 , pH 8.0, 25؇C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution. The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.The biological coordination chemistry of zinc is particularly rich in structural motifs (1), apparently reflecting the numerous functions and general importance of this element in biology (2). One particular motif is that found in biological zinc clusters (3), polynuclear complexes in which zinc is coordinated exclusively to thiolate sulfurs of cysteine residues. Their prominent structural feature includes both bridging and terminal cysteine ligands. Each zinc atom resides in a tetrahedral coordination environment and is apparently unable to accommodate additional ligands. How this coordination relates to biological function is unknown. We will address this question with reference to metallothionein (MT), a bilobal protein harboring two of these clusters.Tetranuclear and trinuclear zinc clusters ( Fig. 1 A and B) are located in two separate domains of mammalian MTs (4, 5). The three-metal cluster in the N-terminal ␤-domain has 6 terminal and 3 bridging cysteine ligands, whereas the four-metal cluster in the C-terminal ␣-domain has 11 cysteines, 6 of the terminal type and 5 that form bridges. As a consequence, all 20 cysteines of MT are involved in the binding of seven zinc atoms. Their unparalleled metal͞ligand ratio makes these biological zinc cluste...

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“…As many as 14 metal atoms (13 cadmium and one zinc) were detected as bound to a single MT molecule. Palumaa and co-workers 34,35 previously reported the formation of a monomeric MT form containing 13 cadmium ions and inorganic phosphate (Cd 13 -(P i ) 2 -MT). Our results are consistent with these findings, suggesting a great degree of structural adaptability of MT in response to various conditions, such as different protein and metal concentrations.…”

Section: Binding Stoichiometry Of Cadmium and Platinum To Mtmentioning

confidence: 99%

“…The rabbit MT contains aspartic acid, threonine, tyrosine, and serine, which contain carboxyl and hydroxyl groups for metal binding. Palumaa and co-workers 34,35 reported the participation of oxygen and/or nitrogen ligands besides thiolates in the formation of Cd 13 -(P i ) 2 -MT. Recent studies on the interactions of cisplatin with ubiquitin demonstrated that cisplatin could bind to ubiquitin at the sites of methionine and histidine.…”

Section: Binding Stoichiometry Of Cadmium and Platinum To Mtmentioning

confidence: 99%

Direct evidence for co‐binding of cisplatin and cadmium to a native zinc‐ and cadmium‐containing metallothionein

Mandal¹,

Jiang²,

Li³

2003

Applied Organom Chemis

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Cisplatin is widely used to treat a number of cancers, and its covalent binding to DNA is believed to cause cell death; however, the roles of cisplatin-protein interactions in the mechanisms of action, toxicity, and resistance of the drug largely remain to be elucidated. Here, we investigate the interactions of cisplatin and a native rabbit metallothionein (MT), containing 1.4% zinc and 7.9% cadmium, using nanospray tandem quadrupole time-of-flight mass spectrometry (MS) and size-exclusion high-performance liquid chromatography with inductively coupled plasma MS. At near-neutral pH conditions, reactions between cisplatin and MT resulted in the formation of complexes that contained Cd 4 -Pt n -MT (n = 1-7). While zinc was displaced by cisplatin, both platinum and cadmium were bound to the same MT molecule. This is the first report to provide direct evidence for the co-binding of cadmium and platinum to MT, which suggests that the mechanism of the binding of cisplatin to the native MT may not be through the displacement of cadmium as previously proposed. A tandem MS investigation into the binding sites of the platinum and cadmium to MT showed platinum-and cadmium-related fragments, such as (PtS 2 C 2 H 7 N) + and (CdS 3 C 5 H 17 N 2 ) + , demonstrating the platinum-cysteine and cadmium-cysteine binding. In addition, detection of Cd 4 -Pt 7 -MT demonstrated more than ten metals bound to a single MT molecule. This finding was extended to the binding of MT with a five-fold excess of CdCl 2 . As many as 14 metal atoms (13 cadmium and one zinc) were detected bound to a single MT molecule, the complexes being Cd x -Zn-MT (x = 5-13). The high binding capacity of MT for cadmium and platinum is consistent with the role of MT in reduction of metal toxicity and its involvement in drug resistance.

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Nonoxidative cadmium-dependent dimerization of cadmium-metallothionein from rabbit liver

Cited by 39 publications

References 32 publications

Purification and Characterisation of Recombinant Sea Urchin Metallothionein Expressed in Escherichia Coli

Purification and Characterisation of Recombinant Sea Urchin Metallothionein Expressed in Escherichia Coli

Coordination dynamics of biological zinc “clusters” in metallothioneins and in the DNA-binding domain of the transcription factor Gal4

Direct evidence for co‐binding of cisplatin and cadmium to a native zinc‐ and cadmium‐containing metallothionein

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