The MNK (
Menkes disease protein; ATP7A) is a major
copper- transporting
P-type ATPase involved in the delivery of
copper to cuproenzymes in the secretory pathway and the efflux of excess
copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in
Menkes disease, a fatal neurodegenerative
copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of
copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal
Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic
detergent DDM (
n-dodecyl beta-D-maltopyranoside). Formation of the classical
vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 microM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent
ATPase activity of MNK (K0.5=0.6 microM; h=5.0). The purified MNK demonstrated active
ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean
asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian
copper-transporting
P-type ATPase associated with a human disease.