{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T05:58:58Z","timestamp":1791179938545,"version":"4.1.0"},"reference-count":58,"publisher":"Portland Press Ltd.","issue":"2","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2007,1,15]]},"abstract":"<jats:p>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 \u03b2-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7\u00a0\u03bcM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6\u00a0\u03bcM; 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.<\/jats:p>","DOI":"10.1042\/bj20060924","type":"journal-article","created":{"date-parts":[[2007,1,4]],"date-time":"2007-01-04T07:38:33Z","timestamp":1167896313000},"page":"569-579","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":34,"title":["Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)"],"prefix":"10.1042","volume":"401","author":[{"given":"Ya\u00a0Hui","family":"Hung","sequence":"first","affiliation":[{"name":"Department of Genetics, University of Melbourne, VIC 3010, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meredith\u00a0J.","family":"Layton","sequence":"additional","affiliation":[{"name":"Joint Proteomics Laboratory, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3050, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ilia","family":"Voskoboinik","sequence":"additional","affiliation":[{"name":"Department of Genetics, University of Melbourne, VIC 3010, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julian\u00a0F.\u00a0B.","family":"Mercer","sequence":"additional","affiliation":[{"name":"Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, VIC 3125, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James","family":"Camakaris","sequence":"additional","affiliation":[{"name":"Department of Genetics, University of Melbourne, VIC 3010, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2006,12,21]]},"reference":[{"key":"2021112213243353000_B1","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1038\/ng0193-14","article-title":"Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein","volume":"3","author":"Chelly","year":"1993","journal-title":"Nat. Genet."},{"key":"2021112213243353000_B2","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1038\/ng0193-20","article-title":"Isolation of a partial candidate gene for Menkes disease by positional cloning","volume":"3","author":"Mercer","year":"1993","journal-title":"Nat. Genet."},{"key":"2021112213243353000_B3","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1038\/ng0193-7","article-title":"Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase","volume":"3","author":"Vulpe","year":"1993","journal-title":"Nat. Genet."},{"key":"2021112213243353000_B4","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1542\/peds.50.2.188","article-title":"Menkes's kinky hair syndrome. An inherited defect in copper absorption with widespread effects","volume":"50","author":"Danks","year":"1972","journal-title":"Pediatrics"},{"key":"2021112213243353000_B5","first-page":"764","article-title":"A sex-linked recessive disorder with retardation of growth and peculiar hair, and focal cerebral and cerebellar degeneration","volume":"29","author":"Menkes","year":"1962","journal-title":"Pediatrics"},{"key":"2021112213243353000_B6","doi-asserted-by":"crossref","first-page":"6084","DOI":"10.1002\/j.1460-2075.1996.tb00997.x","article-title":"Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking","volume":"15","author":"Petris","year":"1996","journal-title":"EMBO J."},{"key":"2021112213243353000_B7","doi-asserted-by":"crossref","first-page":"C1463","DOI":"10.1152\/ajpcell.00179.2004","article-title":"Signals regulating trafficking of Menkes (MNK; ATP7A) copper-translocating P-type ATPase in polarized MDCK cells","volume":"287","author":"Greenough","year":"2004","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"2021112213243353000_B8","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1523\/JNEUROSCI.3699-04.2005","article-title":"NMDA receptor activation mediates copper homeostasis in hippocampal neurons","volume":"25","author":"Schlief","year":"2005","journal-title":"J. Neurosci."},{"key":"2021112213243353000_B9","first-page":"6559","article-title":"Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper","volume":"62","author":"Katano","year":"2002","journal-title":"Cancer Res."},{"key":"2021112213243353000_B10","doi-asserted-by":"crossref","first-page":"1607","DOI":"10.1016\/j.jinorgbio.2004.05.006","article-title":"The role of copper transporters in the development of resistance to Pt drugs","volume":"98","author":"Safaei","year":"2004","journal-title":"J. Inorg. Biochem."},{"key":"2021112213243353000_B11","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.critrevonc.2004.09.007","article-title":"Copper transporters regulate the cellular pharmacology and sensitivity to Pt drugs","volume":"53","author":"Safaei","year":"2005","journal-title":"Crit. Rev. Oncol. Hematol."},{"key":"2021112213243353000_B12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0304-4157(95)00017-8","article-title":"Structural organization, ion transport, and energy transduction of P-type ATPases","volume":"1286","author":"M\u00f8ller","year":"1996","journal-title":"Biochim. Biophys. Acta"},{"key":"2021112213243353000_B13","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1007\/PL00006286","article-title":"Evolution of substrate specificities in the P-type ATPase superfamily","volume":"46","author":"Axelsen","year":"1998","journal-title":"J. Mol. Evol."},{"key":"2021112213243353000_B14","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1016\/S0968-0004(96)20016-7","article-title":"CPx-type ATPases: a class of P-type ATPases that pump heavy metals","volume":"21","author":"Solioz","year":"1996","journal-title":"Trends Biochem. Sci."},{"key":"2021112213243353000_B15","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1038\/35015017","article-title":"Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution","volume":"405","author":"Toyoshima","year":"2000","journal-title":"Nature"},{"key":"2021112213243353000_B16","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1038\/nature00944","article-title":"Structural changes in the calcium pump accompanying the dissociation of calcium","volume":"418","author":"Toyoshima","year":"2002","journal-title":"Nature"},{"key":"2021112213243353000_B17","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1038\/nature02981","article-title":"Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues","volume":"432","author":"Toyoshima","year":"2004","journal-title":"Nature"},{"key":"2021112213243353000_B18","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1038\/nature02680","article-title":"Crystal structure of the calcium pump with a bound ATP analogue","volume":"430","author":"Toyoshima","year":"2004","journal-title":"Nature"},{"key":"2021112213243353000_B19","doi-asserted-by":"crossref","first-page":"1672","DOI":"10.1126\/science.1099366","article-title":"Phosphoryl transfer and calcium ion occlusion in the calcium pump","volume":"304","author":"S\u00f8rensen","year":"2004","journal-title":"Science"},{"key":"2021112213243353000_B20","doi-asserted-by":"crossref","first-page":"28620","DOI":"10.1074\/jbc.M103532200","article-title":"The regulation of catalytic activity of the Menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites","volume":"276","author":"Voskoboinik","year":"2001","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B21","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/S0014-5793(97)00770-9","article-title":"Acylphosphate formation by the Menkes copper ATPase","volume":"412","author":"Solioz","year":"1997","journal-title":"FEBS Lett."},{"key":"2021112213243353000_B22","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/S0014-5793(98)01059-X","article-title":"ATP-dependent copper transport by the Menkes protein in membrane vesicles isolated from cultured Chinese hamster ovary cells","volume":"435","author":"Voskoboinik","year":"1998","journal-title":"FEBS Lett."},{"key":"2021112213243353000_B23","doi-asserted-by":"crossref","first-page":"22008","DOI":"10.1074\/jbc.274.31.22008","article-title":"Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells","volume":"274","author":"Voskoboinik","year":"1999","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B24","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1006\/plas.1997.1334","article-title":"Eukaryotic expression vectors that replicate to low copy number in bacteria: transient expression of the Menkes protein","volume":"39","author":"La\u00a0Fontaine","year":"1998","journal-title":"Plasmid"},{"key":"2021112213243353000_B25","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/0378-1119(91)90366-J","article-title":"Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli","volume":"100","author":"Wang","year":"1991","journal-title":"Gene"},{"key":"2021112213243353000_B26","doi-asserted-by":"crossref","first-page":"22672","DOI":"10.1016\/S0021-9258(17)31698-8","article-title":"Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange","volume":"269","author":"Porfiri","year":"1994","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B27","article-title":"Baculovirus Expression Vectors: A Laboratory Manual","author":"O'Reilly","year":"1994"},{"key":"2021112213243353000_B28","article-title":"Antibodies: A Laboratory Manual","author":"Harlowe","year":"1988"},{"key":"2021112213243353000_B29","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/S0076-6879(03)72004-7","article-title":"Reconstitution of membrane proteins into liposomes","volume":"372","author":"Rigaud","year":"2003","journal-title":"Methods Enzymol."},{"key":"2021112213243353000_B30","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1006\/jsbi.1997.3848","article-title":"Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins","volume":"118","author":"Rigaud","year":"1997","journal-title":"J. Struct. Biol."},{"key":"2021112213243353000_B31","doi-asserted-by":"crossref","first-page":"2117","DOI":"10.1093\/hmg\/4.11.2117","article-title":"Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux","volume":"4","author":"Camakaris","year":"1995","journal-title":"Hum. Mol. Genet."},{"key":"2021112213243353000_B32","doi-asserted-by":"crossref","first-page":"11170","DOI":"10.1074\/jbc.274.16.11170","article-title":"The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein","volume":"274","author":"Strausak","year":"1999","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B33","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1006\/bbrc.2000.4176","article-title":"Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae","volume":"280","author":"Wunderli-Ye","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"2021112213243353000_B34","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/0003-2697(79)90115-5","article-title":"An improved assay for nanomole amounts of inorganic phosphate","volume":"100","author":"Lanzetta","year":"1979","journal-title":"Anal. Biochem."},{"key":"2021112213243353000_B35","doi-asserted-by":"crossref","first-page":"15872","DOI":"10.1074\/jbc.272.25.15872","article-title":"Functional co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation","volume":"272","author":"Autry","year":"1997","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B36","doi-asserted-by":"crossref","first-page":"1470","DOI":"10.1016\/S0021-9258(18)54099-0","article-title":"Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells","volume":"268","author":"DeTomaso","year":"1993","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B37","doi-asserted-by":"crossref","first-page":"976","DOI":"10.1074\/jbc.M109368200","article-title":"Functional properties of the copper-transporting ATPase ATP7B (the Wilson's disease protein) expressed in insect cells","volume":"277","author":"Tsivkovskii","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B38","doi-asserted-by":"crossref","first-page":"652","DOI":"10.1073\/pnas.97.2.652","article-title":"CopA: an Escherichia coli Cu(I)-translocating P-type ATPase","volume":"97","author":"Rensing","year":"2000","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2021112213243353000_B39","doi-asserted-by":"crossref","first-page":"9955","DOI":"10.1016\/S0021-9258(19)86652-8","article-title":"Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column","volume":"254","author":"Niggli","year":"1979","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B40","doi-asserted-by":"crossref","first-page":"3873","DOI":"10.1074\/jbc.275.6.3873","article-title":"The ATP hydrolytic activity of purified ZntA, a Pb(II)\/Cd(II)\/Zn(II)-translocating ATPase from Escherichia coli","volume":"275","author":"Sharma","year":"2000","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B41","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1016\/0005-2736(92)90415-I","article-title":"Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents","volume":"1107","author":"Levy","year":"1992","journal-title":"Biochim. Biophys. Acta"},{"key":"2021112213243353000_B42","doi-asserted-by":"crossref","first-page":"4907","DOI":"10.1016\/S0021-9258(18)83677-8","article-title":"Detergent structure and associated lipid as determinants in the stabilization of solubilized Ca2+-ATPase from sarcoplasmic reticulum","volume":"264","author":"Lund","year":"1989","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B43","doi-asserted-by":"crossref","first-page":"9217","DOI":"10.1074\/jbc.270.16.9217","article-title":"Copper and silver transport by CopB-ATPase in membrane vesicles of Enterococcus hirae","volume":"270","author":"Solioz","year":"1995","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B44","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1016\/S0021-9258(17)43707-0","article-title":"Controlled proteolysis of the purified Ca2+-ATPase of the erythrocyte membrane. A correlation between the structure and the function of the enzyme","volume":"259","author":"Zurini","year":"1984","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B45","doi-asserted-by":"crossref","first-page":"7201","DOI":"10.1074\/jbc.M109964200","article-title":"Characterization of a thermophilic P-type Ag+\/Cu+-ATPase from the extremophile Archaeoglobus fulgidus","volume":"277","author":"Mandal","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B46","doi-asserted-by":"crossref","first-page":"33279","DOI":"10.1074\/jbc.272.52.33279","article-title":"Expression, purification, and metal binding properties of the N-terminal domain from the Wilson disease putative copper-transporting ATPase (ATP7B)","volume":"272","author":"DiDonato","year":"1997","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B47","doi-asserted-by":"crossref","first-page":"13409","DOI":"10.1074\/jbc.M111649200","article-title":"Zinc binding to the NH2-terminal domain of the Wilson disease copper-transporting ATPase: implications for in vivo metal ion-mediated regulation of ATPase activity","volume":"277","author":"DiDonato","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B48","doi-asserted-by":"crossref","first-page":"40858","DOI":"10.1074\/jbc.M107455200","article-title":"Functional analysis of chimeric proteins of the Wilson Cu(I)-ATPase (ATP7B) and ZntA, a Pb(II)\/Zn(II)\/Cd(II)-ATPase from Escherichia coli","volume":"276","author":"Hou","year":"2001","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B49","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1021\/ar0300118","article-title":"Understanding how cells allocate metals using metal sensors and metallochaperones","volume":"38","author":"Tottey","year":"2005","journal-title":"Acc. Chem. Res."},{"key":"2021112213243353000_B50","doi-asserted-by":"crossref","first-page":"3589","DOI":"10.1016\/j.febslet.2005.05.042","article-title":"Purification and functional reconstitution of the human Wilson copper ATPase, ATP7B","volume":"579","author":"Portmann","year":"2005","journal-title":"FEBS Lett."},{"key":"2021112213243353000_B51","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1073\/pnas.0336230100","article-title":"Essential role for Atox1 in the copper-mediated intracellular trafficking of the Menkes ATPase","volume":"100","author":"Hamza","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2021112213243353000_B52","doi-asserted-by":"crossref","first-page":"27953","DOI":"10.1074\/jbc.M203845200","article-title":"Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the Wilson's disease protein and regulates its catalytic activity","volume":"277","author":"Walker","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B53","doi-asserted-by":"crossref","first-page":"9640","DOI":"10.1074\/jbc.M413840200","article-title":"The copper-transporting ATPases, Menkes and Wilson disease proteins, have distinct roles in adult and developing cerebellum","volume":"280","author":"Barnes","year":"2005","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B54","doi-asserted-by":"crossref","first-page":"46987","DOI":"10.1074\/jbc.M208490200","article-title":"Biochemical characterization of CopA, the Escherichia coli Cu(I)-translocating P-type ATPase","volume":"277","author":"Fan","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B55","doi-asserted-by":"crossref","first-page":"6857","DOI":"10.1021\/bi000015+","article-title":"Stoichiometry of complex formation between copper(I) and the N-terminal domain of the Menkes protein","volume":"39","author":"Cobine","year":"2000","journal-title":"Biochemistry"},{"key":"2021112213243353000_B56","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/S0167-4838(99)00161-2","article-title":"Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein","volume":"1434","author":"Jensen","year":"1999","journal-title":"Biochim. Biophys. Acta"},{"key":"2021112213243353000_B57","doi-asserted-by":"crossref","first-page":"46736","DOI":"10.1074\/jbc.M208864200","article-title":"Copper-regulated trafficking of the Menkes disease copper ATPase is associated with formation of a phosphorylated catalytic intermediate","volume":"277","author":"Petris","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112213243353000_B58","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1016\/S0006-291X(03)00010-X","article-title":"Mutational analysis of the Menkes copper P-type ATPase (ATP7A)","volume":"301","author":"Voskoboinik","year":"2003","journal-title":"Biochem. Biophys. Res. Commun."}],"container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/401\/2\/569\/647456\/bj4010569.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/401\/2\/569\/647456\/bj4010569.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,2,9]],"date-time":"2024-02-09T19:32:51Z","timestamp":1707507171000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/401\/2\/569\/42036\/Purification-and-membrane-reconstitution-of"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2006,12,21]]},"references-count":58,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2007,1,15]]}},"URL":"https:\/\/doi.org\/10.1042\/bj20060924","relation":{},"ISSN":["0264-6021","1470-8728"],"issn-type":[{"value":"0264-6021","type":"print"},{"value":"1470-8728","type":"electronic"}],"subject":[],"published":{"date-parts":[[2006,12,21]]}}}