Ion selectivity of the cytoplasmic binding sites of the Na,K-ATPase: I. Sodium binding is associated with a conformational rearrangement

被引:53
|
作者
Schneeberger, A [1 ]
Apell, HJ [1 ]
机构
[1] Univ Konstanz, Dept Biol, D-78457 Constance, Germany
来源
JOURNAL OF MEMBRANE BIOLOGY | 1999年 / 168卷 / 03期
关键词
Na; K-ATPase; cytoplasmic ion binding; electrochromic fluorescent dye; FITC; ion transport; energy transduction mechanism;
D O I
10.1007/s002329900511
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
To investigate Na+ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na+-titration experiments were performed using two fluorescent dyes, RH421(1) and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na+ in the presence of various Mg2+ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na+ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na+ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na+ ions and followed by the conformational transition E-1/E-2 of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na+ ions are bound.
引用
收藏
页码:221 / 228
页数:8
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