The proposal focuses on questions relating to the characterization, structure and formation of metal-thiolate clusters in the 3 subclasses of metallothionein (MT), i.e. polypeptides homologous to mammalian MT, cysteinerich polypeptides only distantly related to MT, and short cysteine-peptides of structure (gamma-Glu-Cys)n- Gly. The MT family molecules is of interest in that they clearly represent a major mechanism of metal ion detoxification in living species. The Y-Glu peptide subclass is a major detoxification system in species not expressing the MT polypeptide. There is suggestive evidence that mammalian MT also functions in some aspect of Cu and Zn ion homeostasis. Three areas of studies will be emphasized. One area of investigation is the structure of Cu(I) clusters in the MT subclasses. Cu(I) ions appear to bind to MT in a tertiary fold distinct from that of Zn or Cd ions. If mammalian MT functions in Cu and Zn homeostasis, it is important to understand the structural differences between CuMT and ZnMT. Structural studies on several different CuMTs will be carried out. Cu binding to MT will be studied to determine whether Cu(I) is capable of associating with MT in either trigonal or tetrahedral geometries. Clusters formed by the MT polypeptide will be compared to clusters formed by the MT gamma-Glu peptides. The role of acid- labile sulfide ions in the Cd-gamma-Glu peptide complex will be studied to determine its structural role in the metal-thiolate cluster. Secondly, we propose to investigate the mechanism by which Cd(II) stimulates Y-Glu peptide synthesis by attempting to duplicate peptide synthesis in vitro. Demonstration of an activity will provide an assay for purification of the putative metal- regulated enzyme. Thirdly, we plan to characterize MT present in serum. We hypothesize that serum MT represents a unique secretory polypeptide unlike the well-known cellular MTs. Demonstration of a secretory MT would be indicative of a novel cellular metal efflux pathway.
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