Occurrence of Cu-ATPase in Dictyostelium: possible role in resistance to copper

Dictyostelium discoideum amoebae showed an uncommon resistance to Cu(2+), as pointed out through cell growth rate (EC(50) = 469 +/- 30 microM) and the neutral red cytotoxicity assay (EC(50) = 334 +/- 45 microM). Although no evidence of Cu-inducible metallothionein was found, Cu-dependent ATPase activity was cytochemically detected on pelletted, resin-embedded amoebae. This activity required Cu(2+) in the incubation medium, was sensitive to TPEN, vanadate and temperature, and showed dose-dependent increase after exposure of amoebae to 10-500 microM Cu(2+) for 7 days. Accordingly, immunofluorescence and Western blotting revealed the occurrence of a Cu-inducible, putative homologue of human Menkes (MNK) Cu-P-type ATPase. To verify if Cu-ATPase is involved in copper resistance, amoebae were exposed to low concentrations of Cu(2+) and vanadate followed by the neutral red assay. Exposure to either treatment showed no effect, while a combination caused a dramatic increase of Cu toxicity, possibly depending on Cu-ATPase inhibition.