title: Order of metal binding in metallothionein.
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.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002104:Cadmium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008668:Metallothionein / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001059:Apoproteins; D002104:Cadmium; D008668:Metallothionein; D015032:Zinc