FUNCTIONAL-ANALYSIS OF A GENETIC-DEFECT OF COPPER TRANSPORT (MENKES DISEASE) IN DIFFERENT CELL-LINES

To define the function of the Cu-transporting ATPase in Menkes disease , Menkes and normal fibroblasts were incubated with Cu-67 before and a fter brief exposure to -SH reagents, p-chloromercuribenzoate (PCMB) an d dithiothreitol (DTT). Accumulation and retention were compared among these cells, BeWo cells, and rat C-6 glioma cells similarly treated. The Michaelis constant for influx of Cu-67 into normal and Menkes fibr oblasts was practically the same (0.21 +/- 0.07 vs. 0.24 +/- 0.06 mu M ). The PCMB treatment stimulated Cu-67 accumulation in C-6 cells, inhi bited accumulation in normal and Menkes fibroblasts, and did not affec t BeWo cells. DTT stimulated Cu-67 uptake in all cells but BeWo cells. DTT treatment after PCMB further enhanced Cu-67 accumulation in norma l fibroblasts and C-6 cells but had no enhancing effect on Menkes fibr oblasts or BeWo cells. Menkes fibroblasts and BeWo cells released Cu-6 7 at rates considerably slower than normal fibroblasts (0.06 and 0.09 vs. 0.22%/min, respectively). The PCMB blocked Cu-67 release from norm al fibroblasts but did not affect Menkes fibroblasts or BeWo cells. Re verse transcription-polymerase chain reaction analysis of total RNA fr om BeWo cells failed to show a predicted 943-base pair fragment repres enting a partial transcript of the Menkes factor. The fragment was pre sent in extracts from normal fibroblasts. We conclude that the mechani sm underlying Cu homeostasis varies among different cell types. As exe mplified by BeWo and Menkes cells, failure to efflux Cu ions may be li nked with the failure to express a functional Cu-transporting ATPase, namely, the Menkes protein.