Copper uptake by cultured trophoblast cells isolated from human term placenta

This paper has examined copper uptake from CuHis2 complexes by cytotrophoblast cells isolated from term human placenta. Uptake is time-dependent, reaching equilibrium after about 90 min, and saturable, with a calculated apparent Km of 0.174 +/- 0.061 microM and Vmax, measured over 30 min, of 0.721 +/- 0.092 pmol/min/micrograms DNA. To determine whether ATP was required for uptake, cells were incubated with inhibitors of glycolysis (iodoacetate) and the TCA cycle (sodium azide and cyanide). Iodoacetate and sodium azide had no effect on uptake, but cyanide decreased the initial rate of uptake. This effect was due to copper binding to the inhibitor and decreasing the effective substrate concentration rather than inhibition of uptake through ATP depletion. Ouabain and monensin had no effect, showing that neither the Na+ gradient nor endocytosis were involved in uptake. The monovalent ion chelator, bathocuproine sulphonate, had no effect on uptake but buthionine sulfoximine, an inhibitor of glutathione synthesis, did decrease both the rate of uptake and equilibrium copper levels, suggesting that copper may bind to glutathione within the cell. The data show that copper is taken up by a passive carrier-mediated transporter and, following uptake, binds to glutathione within the cell.