摘要：The isoforms of rat liver apo-metallothionein (MT) were reconstituted in vitro with Cd and Zn ions to study the order of binding of the seven metal sites. Reconstitution with seven Cd ions resulted in a metalloprotein similar to induced Cd,Zn-MT by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin and the pH-dependent release of Cd. Proteolytic digestion of MT reconstituted with sub-optimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in cluster A. Upon saturation of the four sites in cluster A, binding occurs in the three metal center, cluster B. Samples reconstituted with one to four Cd or Zn ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain encompassing the four metal center. Samples renatured with five to seven Cd ions per MT molecule showed decreasing quantities of alpha fragment and increasing amounts of nativelike MT. The binding process in each domain is cooperative. Reconstitution of apo-MT with two Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4-alpha cluster. Likewise, when Cd5-renatured MT was digested with subtilisin, 30% of the molecules were identified as Cd7-MT with the remainder as Cd4-alpha fragment. Full text Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (809K), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References . 129 130 131 132 133
Loading...
