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Role of glutathione in intracellular copper transport
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Organisms have harnessed the unique chemistry of copper for a variety of purposes. However, that same chemistry makes this essential metal toxic at elevated concentrations. Glutathione (L-7-glutamyl-L-cysteinyl-glycine; GSH), a tripeptide, is thought to play a crucial role in the regulation of this reactive ion. Using reversed phase HPLC, we have identified a glutathione-Cu complex in the acid-soluble fraction of cell extracts from human BeWo cells following treatment with 67CUC12. 67Cu coeluted with oxidized glutathione (GSSG) on the HPLC column suggesting its identity to be a GSSG-CU or GS-CU-SG complex. The stoichiometry of glutathione-Cu complex was sought by uv titration on the spectrophotometer. Absorbance changed because of a Cu to thiolate charge transfer complex and reached its maximum at 260 nm wavelength. When we used degassed water (pH 7.0) as the solvent for GSH and CuCI2, the stoichiometry showed that the ratio of GSH/Cu was 2:1, while 10 mM HEPES/50 mM NaCl buffer (pH 7.4) as solvent produced a ratio of 1: 1. This means that both GSH and GSSG form complexes with Cu. These observations suggest under different circumstances, even with different cell types, we can get different fon-ns of the glutathione-Cu complex. A time study showed that when human BeWo cells were incubated at 370C for various times up to 3 hr, the total cell uptake of 67Cu increased with incubation time, as did the acid-soluble 67Cu and glutathione-67Cu. The percentage of glutathione-Cu in the total cell uptake was more than 50% with these different incubation times. These data suggest that in human BeWo cells, glutathione is probably the first stable carrier to transport Cu from the cell membrane to the inside of the cells, and that the Preexisting pool of glutathione is sufficient to chelate Cu at 50 rtm as long as 180 min before the complex releases Cu to other ligands or organelles in the cell. Evidence obtained from this study suggests that Zn does not form a stable complex with glutathione. Antagonistic effects caused by Zn are on the transport of Cu through the cell membrane and not with glutathione. These studies suggest that glutathione is an important and first ligand of intracellular Cu, which fortns a complex with Cu in vivo. In human BeWo cells, it is GSSG which forms a complex with Cu. However, further research still needs to be done about the chemical identity of this complex.
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Ha, Chenxiang (1994). Role of glutathione in intracellular copper transport. Master's thesis, Texas A&M University. Available electronically from
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