Nucleotide-binding kinetics of Na,K-ATPase: Cation dependence
Nucleotide-binding kinetics of Na,K-ATPase: Cation dependence
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DOI：
10.1021/bi035707n
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发表时间：
2004-04-13
期刊：
影响因子：
2.9
通讯作者：
Esmann, M
中科院分区：
文献类型：
--
作者：
Fedosova, NU;Esmann, M
研究了Na，K-ATP酶对ADP的亲和力与培养基中存在的阳离子（其性质和浓度）之间的相关性。在低离子强度（I约为1 mM）的缓冲液中，未观察到高亲和力ADP结合，而逐步增加所添加的阳离子（Na+、Tris（+）、咪唑（+）、N-甲基葡糖胺（+）、胆碱（+））的浓度诱导ADP亲和力增加。对于所有测试的阳离子，该效应在30-50 mM下完全饱和。在Na+、Tris（+）或咪唑（+）存在下，ADP的最大亲和力略高于N-甲基葡糖胺（+）或胆碱（+）存在下（平衡解离常数K-d 0.2-0.3 vs 0.7 μ M）。在Na+或Tris（+）存在下，ADP与酶复合物的解离速率相似，这意味着核苷酸结合酶构象的同一性，因此将其归属于E-1。与K+竞争的能力清楚地将Na+与其他阳离子区分开来，这与ADP结合E-1构象诱导中运输位点的单独参与相反。由于阳离子在诱导高ADP亲和力的模式上是相似的，但它们在与K+竞争的能力上表现出明显的差异，因此它们的作用不能在仅具有一种类型的阳离子结合位点的任何方案中组合。我们认为，对核苷酸的高亲和力是由蛋白质或脂质内的阳离子相互作用引起的，并且这些核苷酸结构域相关的位点与仅结合Na+或K+的转运位点共存。
Correlation between the Na,K-ATPase affinity to ADP and the cation (its nature and concentration) present in the medium was investigated. In buffer with low ionic strength (I approximate to 1 mM) high-affinity ADP binding was not observed, while a stepwise increase in the concentrations of added cation (Na+, Tris(+), imidazole(+), N-methylglucamine(+), choline(+)) induced an increase in the ADP affinity. The effect was fully saturated at 30-50 mM for all of the cations tested. The maximal affinity for ADP was slightly higher in the presence of Na+, Tris(+), or imidazole(+) than in the presence of N-methylglucamine(+) or choline(+) (equilibrium dissociation constant K-d 0.2-0.3 vs 0.7 muM). The ADP dissociation rates from its complex with enzyme in the presence of Na+ or Tris(+) were similar, implying identity of the nucleotide-binding enzyme conformations, which therefore are assigned to E-1. The ability to compete with K+ clearly distinguished Na+ from other cations, which speaks against the sole involvement of the transport sites in the induction of the ADP-binding E-1 conformation. Since the cations are similar in their mode of induction of the high ADP affinity but they demonstrate a pronounced difference in ability to compete with K+, their effects cannot be combined within any scheme with only one type of cation-binding sites. We suggest that the high affinity toward nucleotide is induced by cation interactions within the protein or lipid and that these nucleotide-domain-related sites coexist with the transport sites, which bind only Na+ or K+.