Functional analysis of a genetic defect of copper transport (
menkes disease) in different cell lines.
Qian, Yongchang, Evelyn Tiffany-Castiglioni, Edward D. Harris.
Department of Biochemistry and Biophysics, Texas A&M University,
College Station, TX 77843, Department of Veterinary Anatomy and
Public Health, Texas A&M University, College Station, TX 77843
APStracts 3:0041C, 1996.
To define the function of the Cu-transporting ATPase in Menkes
disease, Menkes and normal fibroblasts were incubated with 67Cu
before and after brief exposure to -SH reagents, p
-chloromercuribenzoate (p-CMB) and dithiothreitol (DTT). Accumulation
and retention were compared between these cells, BeWo cells, and rat
C6 glioma cells similarly treated. The Michaelis constant (Km) for
influx of 67Cu into normal and Menkes fibroblasts was practically the
same (0.21+/-0.07 [mu]M vs 0.24+/-0.06 [mu]M). p-CMB treatment
stimulated 67Cu accumulation in C6 cells, inhibited accumulation in
normal and Menkes fibroblasts, and did not affect BeWo cells. DTT
stimulated 67Cu uptake in all cells but BeWo cells. DTT treatment
following p-CMB further enhanced 67Cu accumulation in normal
fibroblasts and C6 cells but had no enhancing effect on Menkes
fibroblasts or BeWo cells. Menkes fibroblasts and BeWo cells released
67Cu at a rates considerably slower than normal fibroblasts (0.06%
min-1 and 0.09% min-1 vs 0.22% min-1, respectively). p-CMB blocked
67Cu release from normal fibroblasts but did not affect Menkes
fibroblasts or BeWo cells. RT-PCR analysis of total RNA from BeWo
cells failed to show a predicted 943 bp fragment representing a
partial transcript of the Menkes factor. The fragment was present in
extracts from normal fibroblasts. We conclude that the mechanism
underlying Cu homeostasis varies among different cell types. As
exemplified by BeWo and Menkes cells, failure to efflux Cu ions may
be linked with the failure to express a functional Cu-transporting
ATPase, viz. the Menkes protein.
Received 13 November 1995; accepted in final form 23 January
1996.
APS Manuscript Number C687-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 8 February 96