Effects of metallothionein on the observed copper distribution in cell extracts

Systematic studies have been undertaken to compare the effects of cell lysis and chromatography conditions on the observed distribution of Cu amongst Cu-binding proteins in cultured cells. The variables included rate of centrifugation, presence or absence of non-ionic detergent, and presence or absence of dithiothreitol. The application of an improved FPLC gel filtration system has permitted us to examine the effects of the addition of exogenous metallothionein (MT) to cell extracts. When the cell extract contains low levels of endogenous MT, the addition of MT in the presence of dithiothreitol causes a shift of copper to the MT peak. High levels of MT can therefore remove copper from other Cu-binding ligands during cell homogenization, hence producing artifactual Cu distribution results. The use of an anaerobic buffer system has greatly reduced the observed level of Cu exchange, and has allowed comparison of Cu distribution in normal cells and cells from patients with Menkes' disease.