Metallothionein (MT) coordinates metal ions in 2 domains which enfold separate metalcysteine polynuclear clusters. Cd and Zn ions bind tetrahedrally to MT with a molar equivalency of 7. The alpha and beta domains of Cd,ZnMT complex 4 and 3 metals, respectively. Cu(I) binds at a molar equivalence of 12 with an apparent domain distribution of 6 Cu(I) each. The coordination complex is proposed to consist of Cu(I) ions ligated trigonally by bridging thiolates. We predict that MT binds metals in 2 configurations with Cd7MT and Cu12MT representing prototypes of the 2 forms. We have preliminary data that the following metals conform to M7MT: Hg(II), Pd(II), Bi(III), Pb(II), In(III), Sb(III), and Os(III). Ag(I) appears to bind as M12MT. We intend to confirm these assignments thereby establishing the 2 state model of coordination chemistry. Structural studies on these 2 coordination states will be performed. If MT participates in different intracellular metabolic processes involving Cu and Zn, distinct structures of CuMT and ZnMT may provide a way for a cell to discriminate between the two. Cluster formation induced by Cd, Zn and Cu occurs by an ordered and cooperative process. Whereas Cd and Zn exhibit initial coordination in the alpha domain, Cu initiates cluster formation in the beta domain. Not all metal ions bind to MT cooperatively. We propose to study what distinguishes cooperative from non-cooperative cluster formation. For metals that bind non-cooperatively, we want to know the steps in the buildup of the polynuclear centers. Although the clusters in MT appear to be relatively independent of each other, it is not clear whether the stoichiometry of one domain is influenced by the conformation of the other. We want to determine whether steric constraints preclude formation of a Zn-type tetrahedral center mixed with a Cu-type trigonal center. We propose to characterize the polymorphic forms of CuMT that exist in different subcellular compartments. It has not been rigorously shown whether Cu and Zn induce a different subpopulation of isoforms in the cytoplasm and lysosomes. ApoMT appears to be a likely candidate to regulate the metal responsiveness of the MT gene. In the proposed scheme, apoMT interacts with promoter sequences upstream from the MT genes thereby repressing transcription. Upon verification of the scheme, we propose to use our MT derivatives and fragments to study the interaction between apoMT and DNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Modified Research Career Development Award (K04)
Project #
5K04ES000147-03
Application #
3072695
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1985-07-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Davis, L E; Kornfeld, M; Mooney, H S et al. (1994) Methylmercury poisoning: long-term clinical, radiological, toxicological, and pathological studies of an affected family. Ann Neurol 35:680-8
Mehra, R K; Garey, J R; Winge, D R (1990) Selective and tandem amplification of a member of the metallothionein gene family in Candida glabrata. J Biol Chem 265:6369-75
Mehra, R K; Garey, J R; Butt, T R et al. (1989) Candida glabrata metallothioneins. Cloning and sequence of the genes and characterization of proteins. J Biol Chem 264:19747-53
Dameron, C T; Smith, B R; Winge, D R (1989) Glutathione-coated cadmium-sulfide crystallites in Candida glabrata. J Biol Chem 264:17355-60
Byrd, J; Berger, R M; McMillin, D R et al. (1988) Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants. J Biol Chem 263:6688-94
Mehra, R K; Tarbet, E B; Gray, W R et al. (1988) Metal-specific synthesis of two metallothioneins and gamma-glutamyl peptides in Candida glabrata. Proc Natl Acad Sci U S A 85:8815-9
Reese, R N; Winge, D R (1988) Sulfide stabilization of the cadmium-gamma-glutamyl peptide complex of Schizosaccharomyces pombe. J Biol Chem 263:12832-5
Reese, R N; Mehra, R K; Tarbet, E B et al. (1988) Studies on the gamma-glutamyl Cu-binding peptide from Schizosaccharomyces pombe. J Biol Chem 263:4186-92
Mehra, R K; Winge, D R (1988) Cu(I) binding to the Schizosaccharomyces pombe gamma-glutamyl peptides varying in chain lengths. Arch Biochem Biophys 265:381-9
Thrower, A R; Byrd, J; Tarbet, E B et al. (1988) Effect of mutation of cysteinyl residues in yeast Cu-metallothionein. J Biol Chem 263:7037-42

Showing the most recent 10 out of 12 publications