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Biochem J. Aug 15, 1989; 262(1): 181–188.
PMCID: PMC1133245

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

Abstract

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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Selected References

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  • Byrd J, Winge DR. Cooperative cluster formation in metallothionein. Arch Biochem Biophys. 1986 Oct;250(1):233–237. [PubMed]
  • Furey WF, Robbins AH, Clancy LL, Winge DR, Wang BC, Stout CD. Crystal structure of Cd,Zn metallothionein. Science. 1986 Feb 14;231(4739):704–710. [PubMed]
  • JOVIN T, CHRAMBACH A, NAUGHTON MA. AN APPARATUS FOR PREPARATIVE TEMPERATURE-REGULATED POLYACRYLAMIDE GEL ELECTROPHORESIS. Anal Biochem. 1964 Nov;9:351–369. [PubMed]
  • KAGI JH, VALLEE BL. Metallothionein: a cadmium and zinc-containign protein from equine renal cortex. II. Physico-chemical properties. J Biol Chem. 1961 Sep;236:2435–2442. [PubMed]
  • Klauser S, Kägi JH, Wilson KJ. Characterization of isoprotein patterns in tissue extracts and isolated samples of metallothioneins by reverse-phase high-pressure liquid chromatography. Biochem J. 1983 Jan 1;209(1):71–80. [PMC free article] [PubMed]
  • Li TY, Kraker AJ, Shaw CF, 3rd, Petering DH. Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydrase. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6334–6338. [PMC free article] [PubMed]
  • Nettesheim DG, Engeseth HR, Otvos JD. Products of metal exchange reactions of metallothionein. Biochemistry. 1985 Nov 19;24(24):6744–6751. [PubMed]
  • Neuhaus D, Wagner G, Vasák M, Kägi JH, Wüthrich K. Systematic application of high-resolution, phase-sensitive two-dimensional 1H-NMR techniques for the identification of the amino-acid-proton spin systems in proteins. Rabbit metallothionein-2. Eur J Biochem. 1985 Sep 2;151(2):257–273. [PubMed]
  • Nicholson JK, Sadler PJ, Vasák M. Probing the reactivity of the zinc and cadmium ions bound to rabbit liver metallothioneins with EDTA. Experientia Suppl. 1987;52:191–201. [PubMed]
  • Nielson KB, Winge DR. Order of metal binding in metallothionein. J Biol Chem. 1983 Nov 10;258(21):13063–13069. [PubMed]
  • Nielson KB, Atkin CL, Winge DR. Distinct metal-binding configurations in metallothionein. J Biol Chem. 1985 May 10;260(9):5342–5350. [PubMed]
  • Otvos JD, Armitage IM. Structure of the metal clusters in rabbit liver metallothionein. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7094–7098. [PMC free article] [PubMed]
  • Petering DH, Krezoski S, Villalobos J, Shaw CF, 3rd, Otvos JD. Cadmium-zinc interactions in the Ehrlich cell: metallothionein and other sites. Experientia Suppl. 1987;52:573–580. [PubMed]
  • Piotrowski JK, Szymańska JA, Mogilnicka EM, Zelazowski AJ. Renal metal binding proteins. Experientia Suppl. 1979;34:363–371. [PubMed]
  • Stillman MJ, Zelazowski AJ. Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes. J Biol Chem. 1988 May 5;263(13):6128–6133. [PubMed]
  • Stillman MJ, Cai W, Zelazowski AJ. Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+. J Biol Chem. 1987 Apr 5;262(10):4538–4548. [PubMed]
  • Stillman MJ, Zelazowski AJ, Gasyna Z. Luminescent Ag12-metallothionein: dependence of emission intensity on silver-thiolate cluster formation. FEBS Lett. 1988 Nov 21;240(1-2):159–162. [PubMed]
  • Winge DR, Miklossy KA. Domain nature of metallothionein. J Biol Chem. 1982 Apr 10;257(7):3471–3476. [PubMed]
  • Zelazowski AJ, Szymańska JA, Witas HW. Purification of low molecular weight metal-binding proteins by preparative polyacrylamide gel electrophoresis: properties of electrophoretically purified rat liver (Cd, Zn) - metallothioneins. Prep Biochem. 1980;10(4):495–505. [PubMed]
  • Zelazowski AJ, Szymanska JA, Law AY, Stillman MJ. Spectroscopic properties of the alpha fragment of metallothionein. J Biol Chem. 1984 Nov 10;259(21):12960–12963. [PubMed]

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