Biochem. J. (2007) 401
(569–579) (Printed in Great Britain)
Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)
Ya Hui HUNG*1, Meredith J. LAYTON†, Ilia VOSKOBOINIK*2, Julian F. B. MERCER‡ and James CAMAKARIS*3
*Department of Genetics, University of Melbourne, VIC 3010, Australia, †Joint Proteomics Laboratory, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3050, Australia, and ‡Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, VIC 3125, Australia
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.
Key words: copper homoeostasis, n-dodecyl b-D-maltopyranoside (DDM), Menkes-disease protein (MNK; ATP7A), P-type ATPase, Sf9 [Spodoptera frugiperda (fall armyworm) 9] cell.
Abbreviations used: Atox1, ATX1 antioxidant protein 1 homologue (yeast); BCS, bathocuproinedisulfonic acid; CHO-K1 cell, Chinese-hamster ovary K1 cell; CMC, critical micelle concentration; DDM, n-dodecyl b-D-maltopyranoside; DTT, dithiothreitol; EE tag, Glu-Glu epitope tag; EE–MNK, EE-tagged MNK; HRP, horseradish peroxidase; LUV, large unilamellar vesicles; MBS, metal binding site; 2ME, 2-mercaptoethanol; MNK, Menkes disease protein; MOI, multiplicity of infection; NEM, N-ethylmaleimide; OG, n-octyl b-D-glucopyranoside; pfu, plaque-forming units; SERCA1a, sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1a; TX100, Triton X-100; WND, Wilson disease protein.
1Present address: Oxidation Disorders Laboratory, The Mental Health Research Institute of Victoria, Parkville, VIC 3052, Australia.
2Present address: Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrew's Place, East Melbourne, VIC 3002, Australia.
3To whom correspondence should be addressed (email j.camakaris@unimelb.edu.au).
Received 19 June 2006/28 September 2006; accepted 29 September 2006
Published as BJ Immediate Publication 29 September 2006, doi:10.1042/BJ20060924
The Biochemical Society, London ©2007
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