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Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

Hung, Ya Hui, Layton, Meredith, Voskoboinik, Ilia, Mercer, Julian and Camakaris, James 2007, Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A), Biochemical journal, vol. 401, no. 2, pp. 569-579, doi: 10.1042/BJ20060924.

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Title Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)
Author(s) Hung, Ya Hui
Layton, Meredith
Voskoboinik, Ilia
Mercer, Julian
Camakaris, James
Journal name Biochemical journal
Volume number 401
Issue number 2
Start page 569
End page 579
Publisher Biochemical Society
Place of publication London, England
Publication date 2007
ISSN 0264-6021
1470-8728
Keyword(s) copper homoeostasis
n-dodecyl β-D-maltopyranoside (DDM)
Menkes-disease protein (MNK;ATP7A)
P-typeATPase
Sf9 [Spodoptera frugiperda (fall armyworm) 9] cell
Summary 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 b-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 µM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 µM; 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.
Language eng
DOI 10.1042/BJ20060924
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
HERDC Research category C1 Refereed article in a scholarly journal
Copyright notice ©2007, Biochemical Society
Persistent URL http://hdl.handle.net/10536/DRO/DU:30007648

Document type: Journal Article
Collection: School of Life and Environmental Sciences
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