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  • Title: Order of metal binding in metallothionein.
    Author: Nielson KB, Winge DR.
    Journal: J Biol Chem; 1983 Nov 10; 258(21):13063-9. PubMed ID: 6630220.
    Abstract:
    Purified isoforms of rat liver apometallothionein were reconstituted in vitro with Cd and Zn ions to study the order of binding of the 7 metal sites in the two separate metal clusters, one containing four metal ions (cluster A) and the other containing three (cluster B). Reconstitution with 7 Cd ions resulted in a metalloprotein similar to induced Cd,Zn-metallothionein by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin, and the pH-dependent release of Cd. Proteolytic digestion of metallothionein reconstituted with suboptimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in the 4-metal center, cluster A. Upon saturation of the 4 sites in cluster A, binding occurs in the 3-metal center, cluster B. Samples reconstituted with 1 to 4 Cd ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain that is known to encompass the 4-metal center. Samples renatured with 5 to 7 Cd ions per metallothionein molecule showed decreasing quantities of alpha fragment and increasing amounts of native-like metallothionein. Similar results were obtained in reconstitution studies with Zn ions. Samples reconstituted with 7 Cd eq followed by incubation with EDTA revealed that cluster B Cd ions were removed initially. The binding process in each domain is cooperative. Reconstitution of apometallothionein with 2 Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4 alpha cluster. Likewise, when Cd5-renatured metallothionein was digested with subtilisin, 30% of the molecules were identified as Cd7 metallothionein with the remainder as Cd4 alpha fragment.
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