Yc. Qian et al., FUNCTIONAL-ANALYSIS OF A GENETIC-DEFECT OF COPPER TRANSPORT (MENKES DISEASE) IN DIFFERENT CELL-LINES, American journal of physiology. Cell physiology, 40(1), 1996, pp. 378-384
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.