Copper-zinc superoxide dismutase and intracellular copper incorporation

Copper deficient chicken aortas were grown in culture to study the metal-dependent mechanism for restoring CuZnSOD activity and delivering copper to the cell. Intracellular incorporation of copper was detected by measuring the activity of CuZnSOD and/or incorporation of radioactive copper into CuZnSOD. Cultures supplemented with CuCl2, albumin-Cu, or ceruloplasmin required 24 h and 0.3 ug Cu/ml medium to fully restore the enzyme. Activation did not occur at 40C or with Cu-EDTA as the supplement. Mn+2, Zn+2 alone or in combination, did not activate or enhance the activation achieved by CuCl2. Cordycepin and cycloheximide added to the medium did not inhibit the activation, supporting the suggestion that copper is activating an apoCuZnSOD. 67Cu-labeled albumin and ceruloplasmin added to the culture were able to incorporate 67Cu into CuZnSOD, as detected by immunoprecipitation of CuZnSOD