Role of copper in the regulation of CU, ZN-superoxide dismutase in human K562 erythroleukemia cells and human fibroblasts

Abstract

Activation of the enzyme CU2Zn2-SUperoxide dismutase (CuZnSOD) by its copper cofactor was studied in K562 erythroleukemia cells and skin fibroblasts. K562 cells were incubated in medium supplemented with 0-50 IIM CUC12 or ZnCI2 for 24 h and extracts from the cells for each treatment were assayed for CuZnSOD activity. The results of the study showed that CuZnSOD activity increased when the extracellular copper concentration exceeded 10 [ ]M, and reached maximal activation when the copper concentration was 50 [ ] M. At this point the activity was about 11.5 times greater than untreated cells. A time study showed that the activation was slow at first but then accelerated, suggesting that in K562 cells, copper may activate a preexisting pool of apoSOD as well as stimulate transcription of CuZnSOD MRNA. In contrast, the addition of zinc had no effect on the basal level of CuZnSOD activity, even at a concentration of 50 gM. Two CuZnSOD MRNA species (0.5 and 0.7 kb) were detected in the K562 cells by Northern analysis. Both increased gradually when the cells were incubated with 50 gM CUC12.The copper content in Menkes' cells was twice the concentration of normal fibroblasts. Menkes' fibroblasts also had about twice the CuZnSOD activity, suggesting that intracellular copper was correlated to the increased CuZnSOD activity. CuZnSOD MRNA in Menkes' fibroblasts was detectable but could not be quantitated, whereas CuZnSOD MRNA in normal fibroblasts could not be seen by our procedure. These studies provide supporting evidence that the CuZnSOD activity, protein, and MRNA are all linked to the intracellular level of copper.