Abnormal copper metabolism in menkes cultured fibroblasts

Abstract

Menkes fibroblast cultures were established and copper metabolism was investigated. Menkes fibroblasts contained four to six times higher concentrations of copper than control cells. In Menkes cells more than 90% of the intracellular copper was present in cytosol (105,000 x g supernatant fraction); in control fibroblasts the corresponding value was about 67%. During cultivation of fibroblasts in medium supplemented with 100 ng/ml of copper (270 ng CuCl₂·2H₂O /ml), the amount of copper increased continuously in Menkes cells, at least up to 4 days, while in control cells in reached a maximum after 24 h, followed by a gradual decrease. When the medium was replaced with one without copper chloride, copper concentrations in Menkes cells returned to the original level in three days, whereas those in control cells returned to the normal level in one day. Using Sephadex G-75 column chromatography of cytosol, two copper-containing peaks were observed (peaks 1 and 3, corresponding to the peaks from rat liver cytosol). Approximately 75% of the copper in the cytosol from Menkes cells was eluted in peak 3. The corresponding copper peak was very small in control cells. Copper peaks 1 and 3 from both cells increased after treating cells with copper chloride and the increase was inhibited by cycloheximide, an inhibitor of protein biosynthesis.

Metallothionein purified from human kidneys was eluted as a single copper-protein from a Sephadex G-75 column in the same fractions as peak 3. SDS polyacrylamide-slab gel electrophoresis of the purified metallo-thionein and the material eluted in peak 3 from Menkes fibroblasts showed single peaks for copper at identical migration distances.