Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)
Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)
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DOI：
10.1042/bj20060924
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发表时间：
2007-01-15
影响因子：
4.1
通讯作者：
Camakaris, James
中科院分区：
文献类型：
--
作者：
Hung, Ya Hui;Layton, Meredith J.;Camakaris, James
MNK（门克斯病蛋白；ATP7A）是一种主要的铜转运p型atp酶，参与在分泌途径中将铜传递给铜原酶和过量铜从肝外组织外排。MNK （ATP7A）基因突变导致Menkes病，一种致命的神经退行性铜缺乏症。目前，由于缺乏活性形式的纯化MNK，无法对MNK进行详细的生化和生物物理分析，以更好地了解其铜转运机制。为了解决这一问题，我们在Sf9 [Spodoptera frugiperda（秋粘虫）9]昆虫细胞中表达了带有n端Glu-Glu标签的人MNK，并在非离子洗涤剂DDM （n-十二烷基β -d -maltopyranoside）存在下，通过抗体亲和层析和大小排斥层析纯化了MNK。Cu(I)激活纯化的MNK形成经典的钒酸盐敏感磷酸酶[EC50 = 0.7 μ M；h （Hill系数）为4.6]。此外，我们报道了MNK中Cu(I)依赖性atp酶活性的首次测量（K-0.5 = 0.6 μ M; h = 5.0）。纯化的MNK在重组成大豆凝集素脂质体时显示出活性的atp依赖载体Cu-64转运。总之，这些数据表明Cu(I)与MNK以合作的方式相互作用，并且在亚微摩尔范围内具有高亲和力。本研究首次提供了纯化的全长哺乳动物铜转座p型atp酶与人类疾病相关的生化表征。
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 mu M; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K-0.5 = 0.6 mu M; h = 5.0). The purified MNK demonstrated active ATP-dependent vectorial Cu-64 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-transponing P-type ATPase associated with a human disease.