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

Dictyostelium discoideum amoebae showed an uncommon resistance to Cu2+, as pointed out through cell growth rate (EC50 = 469 +/- 30 muM) and the neutral red cytotoxicity assay (EC50 334 +/- 45 muM). Although no evidence of Cu-inducible metallothionein was found, Cu-dependent ATPase activity was cytochemically detected on pelletted, resin-embedded amoebae. This activity required Cu2+ in the incubation medium, was sensitive to TPEN, vanadate and temperature, and showed dose-dependent increase after exposure of amoebae to 10-500 muM Cu2+ 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 Cu2+ 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. (C) 2002 Elsevier Science (USA).