Abstract
The copper complex of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone) or CuKTS is reduced and dissociated upon reaction with Ehrlich cells. Titration of the cells with the complex leads to the specific binding of copper to metallothionein with 1 to 1 displacement of its complement of zinc. Under conditions of complete titration of metallothionein, 1.25-2.5 nmol CuKTS/10(7) cells, cellular DNA synthesis is rapidly inhibited but no long term effects on cell proliferation are observed. The kinetics of redistribution of Cu and Zn in Ehrlich cells in culture and in animals were studied after pulse reaction of CuKTS with cells. After exposure of cells to the noncytotoxic concentration of 2.5 nmol of CuKTS/10(7) cells, nonmetallothionein bound copper is lost rapidly from the cells, after which copper in metallothionein decays. New zinc metallothionein is made as soon as exposed cells are placed in culture. New synthesis stops when the level of zinc in metallothionein reaches control levels. A second pulse treatment of cells with CuKTS to displace zinc from metallothionein again stimulates new synthesis of the protein to restore its normal concentration. The kinetics of metal metabolism in Ehrlich cells exposed to 5.5 nmol of CuKTS/10(7) cells, which inhibits cell proliferation, are qualitatively similar except there is a pronounced lag before new zinc metallothionein is synthesized. The Ehrlich ascites tumor in mice responds to CuKTS similarly to cells in culture. It is also shown that cultured Ehrlich cells do not make extra zinc metallothionein in the presence of high levels of ZnCl2, and fail to accumulate copper in the presence of large concentrations of CuCl2.
Highlights
Studied after pulse reaction of CuKTS with cells
2.5 nmol of CuKTS/107 cells, nonmetallothionein The complex is rapidly taken into cells and reductively disbound copperis lost rapidly from thcells, after which sociated [6]
New zinc metallothionein is made as soon as exposed cellsare placed in culture
Summary
Ehrlich cells CuKTS undergoes the following reaction. studied after pulse reaction of CuKTS with cells. The animal experiments contained greater than lo cells and the final supernatant volume was greater than 5 ml This amount molecular weight peak comprising about 80% of this metal and a peak at 10,000, which is metallothionein. At a higher concentration of CuKTS, 5.5 nmol/107cells, copper has completelydisplaced zinc from metallothionein and is accumulating in the other macromolecular band. Calculations of copper/unit amount of protein molecular band of Cu or from metallothionein was assumed in all or thiol in each fraction at theend pointof binding Cu to Mt cases to be first order processes. This was supported by data, which contained several time points for the turnover of Cu from each of these pools. Titration of Ehrlich Cells with CuKTS: Metal Distribution population excluded trypan blue at the conclusion of the and Biological Responses-Ehrlich cells were placed in Eagle's experiment
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