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.
Burlando
Bruno
B
Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale Amedeo Avogadro, Alessandria, 15100, Italy. burlando@unipmn.it
Evangelisti
Valentina
V
Dondero
Francesco
F
Pons
Giovanni
G
Camakaris
Jim
J
Viarengo
Aldo
A
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis; D027682:Cation Transport Proteins; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003300:Copper / Q000494:pharmacology / Q000633:toxicity*; D004023:Dictyostelium / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005455:Fluorescent Antibody Technique; D008668:Metallothionein / Q000096:biosynthesis; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D011993:Recombinant Fusion Proteins; D003300:Copper; D008668:Metallothionein; D000251:Adenosine Triphosphatases