Metal binding in metallothioneins: Competition for cadmium and zinc between chelex-100 and metal binding sites in metallothionein

Cai, Wuhua; Stillman, Martin J.

doi:10.1016/s0020-1693(00)83342-3

Cited by 13 publications

(5 citation statements)

References 15 publications

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“…As a control, Chelex-100 was added to 10 μM Zn 7 -MT alone. Chelex-100 binds free Zn(II) and Cu(I) ions up to a capacity of 22 μg of Zn(II)/mg of Chelex and 19 μg of Cu(I)/mg of Chelex . The control data show that Chelex-100 does not remove Zn(II) bound to metallothionein under these conditions.…”

Section: Resultsmentioning

confidence: 94%

“…Chelex-100 binds free Zn(II) and Cu(I) ions up to a capacity of 22 µg of Zn(II)/mg of Chelex and 19 µg of Cu(I)/mg of Chelex. 40 The control data show that Chelex-100 does not remove Zn(II) bound to metallothionein under these conditions. The data for Zn 7 -MT + 13Cu(I) show that complete replacement of the Zn(II) bound to the protein by the 12 Cu(I) occurs and that there is no free Cu(I) in solution after 5 min.…”

Section: Resultsmentioning

confidence: 95%

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Mobility of Copper in Binding Sites in Rabbit Liver Metallothionein 2

Green

Stillman

1996

Inorg. Chem.

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 95%

Mobility of Copper in Binding Sites in Rabbit Liver Metallothionein 2

Green

Stillman

1996

Inorg. Chem.

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“…The process of replacement was monitored by CD spectroscopy for Cd 7 -MT 6 and emission spectroscopy for Cu 12 -MT . The free Zn(II) ions from Zn 7 -MT were removed by Chelex-100 . Apo-MT 1 was prepared from Zn 7 -MT 1 by passing the protein down a Sephadex G-25 gel column, which had previously been equilibrated with a pH 2 acetic acid solution.…”

Section: Methodsmentioning

confidence: 99%

“…30 The free Zn(II) ions from Zn7-MT were removed by Chelex-100. 31 Apo-MT 1 was prepared from Zn 7-MT 1 by passing the protein down a Sephadex G-25 gel column, which had previously been equilibrated with a pH 2 acetic acid solution. Preparation of Ag 12-MT 1 and Ag17-MT 1 was carried out by addition of Ag(I) as a form of AgNO 3 to apo-MT 1 at pH 3 and 50 °C; metal binding was monitored by the appearance of characteristic bands in the circular dichroism spectrum.…”

Section: Methodsmentioning

confidence: 99%

Sulfur K-Edge EXAFS Studies of Cadmium-, Zinc-, Copper-, and Silver-Rabbit Liver Metallothioneins

Green

Kasrai

et al. 1996

Inorg. Chem.

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The structures of metal-thiolate clusters in Zn(7)-MT, Cd(7)-MT, Cu(12)-MT, Ag(12)-MT, and Ag(17)-MT from rabbit liver have been investigated by sulfur K-edge X-ray absorption spectroscopy (XAS). In addition to providing metal-cysteinyl sulfur bond lengths, the sulfur K-edge EXAFS data provide the first direct evidence for mixtures of bridging and terminal sulfurs in Cu-MT and Ag-MT. The Zn-S and Cd-S bond lengths for tetrahedrally coordinated Zn(4)(SPh)(10)(2-) and Cd(4)(SPh)(10)(2-) compounds obtained from sulfur K-edge EXAFS data are 2.35 +/- 0.03 and 2.52 +/- 0.02 Å, respectively. Zn-S and Cd-S bond distances of 2.34 +/- 0.03 Å for Zn(7)-MT and 2.54 +/- 0.02 Å for Cd(7)-MT, respectively, calculated from sulfur K-edge EXAFS measurements, are consistent with the previously reported results from metal K-edge EXAFS data. Analysis of the sulfur K-edge EXAFS data for Cu(12)-MT indicates that Cu(I) is trigonally coordinated to sulfurs at a distance of 2.25 +/- 0.01 Å. Significant changes in CD spectra observed between Ag(12)-MT 1 and Ag(17)-MT 1 indicate that the modification of the three-dimensional structure occurs when Ag(17)-MT 1 is formed from Ag(12)-MT 1 as Ag(I) is added to the Ag(12)-MT 1. The Ag-S bond distances of 2.45 +/- 0.02 and 2.44 +/- 0.03 Å in Ag(1)(2)-MT 1 and Ag(1)(7)-MT 1, respectively, calculated from the sulfur K-edge EXAFS measurements, lead us to conclude that the Ag(I) in both Ag(1)(2)-MT 1 and Ag(1)(7)-MT 1 is digonally coordinated by thiolates. The number of metals bonded to sulfur in both model compounds and metal-containing metallothioneins is estimated from sulfur K-edge EXAFS measurements to be in the range 1.2-1.7, depending on the fraction of bridging sulfurs present in compounds. Unlike the spectral data recorded during Cu(I) binding, where sharp changes take place past 12 Cu(I), the CD data for Ag-MT 1 show little variation over the entire range of Ag(I):MT molar ratios. This result, established by both low- and high-energy optical methods, suggests that the three-dimensional structure of the metal-binding sites in metallothioneins is strongly influenced by the fraction of bridging sulfur. This analysis is the first to provide direct support for the presence of a clustered Ag-S structure for the Ag(17)-MT 1 species. These data also suggest that the structures in Ag(I) and Cu(I) metallothioneins are probably quite different.

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“…During the addition of Cd21 to Zn7-MT, we would expect that when the Cd exceeded the capacity of the a-domain (4 molar equivalents) the Cd would accumulate in the fl-domain, displacing the Zn (Winge & Miklossy, 1982;Nielsen et al, 1985). From competitive binding studies with EDTA (Li et al, 1980) and immobilized EDTA (Chelex-100) (Cai & Stillman, 1988a), we would expect that the Cd2+ would bind to the fl-domain tighter than it would to the EDTA [Petering et al (1987) report that the stability constant for Cd2+ binding to MT is 102 times greater than for Cd2+ binding to EDTA]. However, our data show that simply incubating Cd7-MT with EDTA and subtilisin for 2 h is sufficient to remove the 3 Cd atoms from the fl-domain.…”

Section: Discussionmentioning

confidence: 99%

Domain-specificity of Cd2+ and Zn2+ binding to rabbit liver metallothionein 2. Metal ion mobility in the formation of Cd4-metallothionein α-fragment

Stillman

Żelazowski

1989

Biochemical Journal

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The yield of the alpha-fragment of rabbit liver metallothionein 2 was used to test the domain-specificity and mobility of Cd2+ and Zn2+ when bound to metallothionein. Increasing molar ratios of Cd2+ were added to either Zn7-metallothionein or the metal-ion-free apo-metallothionein. The enzyme subtilisin was used to digest those parts of the peptide chain that were not bound to Cd2+. Analysis of the digestion products was carried out by separation by polyacrylamide-gel electrophoresis. The chelation agent EDTA was used as a competitive chelator. It was found that the presence of excess EDTA greatly enhances the formation of the Cd4-metallothionein alpha-fragment, and catalyses the complete digestion of all other the metal-ion-containing peptides, so that even Cd7-metallothionein, formed when 7 molar equivalents of Cd2+ are added to Zn7-metallothionein, is digested to the alpha-fragment. These results suggest that the Cd2+ bound in the beta-sites is very labile, much more labile than the kinetics of the off-reaction would suggest. The observation of significant amounts of alpha-fragment on the gels, even when the stoichiometry of the metal ions initially present in the protein should not have resulted in much concentration of Cd4-alpha-fragment clusters, indicates that as the digestion proceeds the metal ions move to sites that form complete clusters and therefore selectively protect that part of the peptide chain from digestion. We also find that rabbit Cd4-metallothionein 2 alpha-fragment stains near to the top of the gel, in complete contrast with the location of rat Cd4-metallothionein 2 alpha-fragment. This difference in the mobilities suggests that the alpha-fragment prepared from rabbit metallothionein 2 is much less negatively charged than the analogous protein fragment prepared from rat liver metallothionein 2.

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Metal binding in metallothioneins: Competition for cadmium and zinc between chelex-100 and metal binding sites in metallothionein

Cited by 13 publications

References 15 publications

Mobility of Copper in Binding Sites in Rabbit Liver Metallothionein 2

Mobility of Copper in Binding Sites in Rabbit Liver Metallothionein 2

Sulfur K-Edge EXAFS Studies of Cadmium-, Zinc-, Copper-, and Silver-Rabbit Liver Metallothioneins

Domain-specificity of Cd2+ and Zn2+ binding to rabbit liver metallothionein 2. Metal ion mobility in the formation of Cd4-metallothionein α-fragment

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