Osmotic stress and viscous retardation of the Na,K-ATPase ion pump
Osmotic stress and viscous retardation of the Na,K-ATPase ion pump
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DOI：
10.1529/biophysj.106.101774
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发表时间：
2008-04-01
影响因子：
3.4
通讯作者：
Marsh, Derek
中科院分区：
文献类型：
--
作者：
Esmann, Mikael;Fedosova, Natalya U.;Marsh, Derek
Na泵（Na，K-ATP酶）在细胞离子稳态中的转运功能涉及细胞质中的核苷酸结合反应和向内和向外的离子结合位点的交替水暴露。一种具有生物活性的非渗透性聚合物（聚乙二醇）和水溶液粘度的调节剂（甘油）被用来探测鲨鱼盐腺膜Na，K-ATP酶的全部和部分酶反应。两者都抑制稳态Na，K-ATP酶和Na-ATP酶活性，而K+依赖性磷酸酶活性几乎不受高达50%的影响。Na，K-ATP酶和Na-ATP酶的活性都与甘油溶液的粘度成反比，其中膜悬浮在甘油溶液中，这与Kramers关于活性位点处的波动与水性环境中的溶剂流动性的强耦合的理论一致。PEG降低了对Tl+（K+的同源物）的亲和力，而甘油在NaCl存在下增加了对核苷酸ATP和ADP的亲和力，但对Tl+的亲和力几乎没有影响。根据对PEG诱导的渗透胁迫的依赖性，Na，K-ATP酶反应的水性活化体积估计为类似于5-6 nm（3）（即，类似于180个水分子），对于Na-ATP酶大约是一半，而对于对硝基苯酚磷酸酶基本上为零。与T1+结合相关的含水水化体积的变化在9 nm 3的区域中。同源Ca-ATP酶的15种晶体结构的分析揭示了在El-核苷酸结合形式和E-2-毒胡萝卜素形式之间PEG不可接近的水空间增加了类似于22 nm（3），表明Na，K-ATP酶的实验活化体积与Ca-ATP酶的主要E-1和E-2构象之间的水合的总体变化相当。
The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K+-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers' theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl+ (a congener for K+), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl+. From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be similar to 5-6 nm(3) (i.e., similar to 180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl+ is in the region of 9 nm3. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of similar to 22 nm(3) between the El-nucleotide bound forms and the E-2-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E-1 and E-2 conformations of the Ca-ATPase.