This proposal sets forth integrated physiochemical and biological studies to elucidate biologically relevant structure-function relationships of the metal binding protein, Metallothionein (Mt). This protein is a normal constituent of mammalian tissues. It is also the one well defined site to which the environmentally important, toxic heavy metal Cd binds as it reacts with cells. Years of study have demonstrated that Mt plays a central role in Zn and Cu metabolism and the biochemical toxicology of Cd. However, the nature of its biological functions and, indeed, chemical properties are ill defined. The overall goal of the proposed research is to understand the metal-ligand chemistry of Mt, its biological functions in Zn and Cd metabolism and how these are interrelated. The four co-investigators are independent scientists in complementary research area: JDO in multinuclear NMR structure-function studies of metalloproteins, CFS in inorganic and bioinorganic studies of metallo-drugs and proteins, SSB in the culture and examination of transport properties of kidney cortical cells, and DHP in the cellular metabolism of essential and toxic metals and bioinorganic studies of metal complexes and metallo-proteins. The author's broad, overlapping interests undergird this proposal and lead to the following specific aims: (I) To understand in detail the ligand substitution chemistry of Zn- and Cd-Mt involving either small ligands competing for the Mt-bound metal or apometallo-proteins. Once these homogenous metallothioneins are understood, to investigate the properties of mixed metal, Cd, Zn-metallothioneins in ligand substitution chemistry. (II) To use the kidney cortical cell model to study the normal functions of Zn-Mt, possibly involving biological ligand substitution reactions. (III) Finally, to study the interactions of Cd with this system as they relate to Zn-metallothionein function in metal donation reactions and to the binding of Cd to other sites in the cell. Integrated experiments are designed to study side by side the chemistry of Mt which occurs in vitro with that which may be occurring in cells. Chemical studies are predicted on and use the detailed understanding of metallothionein cluster structures provided by 113 Cd NMR. They will center on careful spectrophotometric kinetic studies to understand the mechanism of the ligand substitution reactions of metallothioneins. Kidney cortical cells will be used as a cellular model highly relevant to Cd toxicology. To relate the chemical results to cells, kinetics of distribution of Zn and Cd among cell components and compartments will be carried out using radiotracers to follow pools of metal and protein. Compartments will be carried out in conjunction with assessment of effects of Cd on cortical cell function.
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