| Abstract: | Within 24 to 72 hr after hatching, chicks' total aortic SOD activity, cyanide-sensitive (CuZnSOD) and insensitive (MnSOD), rose 3-fold and stayed elevated for 10 days. A copper-deficient diet did not appear to affect the initial rise but promoted a rapid decline in, and ultimately the loss of, detectable activity, within 8-10 days. CuZnSOD activity was restored in about 8 h by a single i.p. injection of 1 mg CuSO₄/kg body weight. Although extracts from 15-day deficient aortas had no detectable CuZnSOD activity they contained as much immunoreactive CuZnSOD protein as extracts from control tissue, suggesting the presence of an apo-enzyme. Copper deficient 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 4°C 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. The transfer of ⁶⁷Cu by ceruloplasmin was inhibited by unlabeled ceruloplasmin; disodium EDTA (0.1 mM) had no effect on the transfer of copper to the enzyme. Newly hatched chicks appear capable of synthesizing active CuZnSOD through the first 2-3 days of growth. As dietary copper becomes limiting, the synthesis of CuZnSOD is inhibited or continues with the production of an apoform. The activation of the enzyme is specific for copper and the transfer of copper into the enzyme structure appears to be the primary event restoring catalytic activity to the enzyme. |
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