K⁺ and Rb⁺ Affinities of the Na,K-ATPase α₁ and α₂ Isozymes: An Application of ICP-MS for Quantification of Na⁺ Pump Kinetics in Myofibers.
K⁺ and Rb⁺ Affinities of the Na,K-ATPase α₁ and α₂ Isozymes: An Application of ICP-MS for Quantification of Na⁺ Pump Kinetics in Myofibers.
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DOI：
10.3390/ijms19092725
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发表时间：
2018-09-12
影响因子：
5.6
通讯作者：
Heiny JA
中科院分区：
文献类型：
--
作者：
Hakimjavadi H;Stiner CA;Radzyukevich TL;Lingrel JB;Norman N;Landero Figueroa JA;Heiny JA
Na，K-ATP酶同工酶的钾亲和力是决定其在骨骼肌中生理作用的重要因素。本研究测量了从WT和遗传改变小鼠（α 1 S/Sα 2 R/R和skα2−/−）获得的完整、分离肌纤维中Na，K-ATP酶α1和α2同工酶的表观K+和Rb+亲和力。它还验证了一种新的方法来量化阳离子在完整的，解离肌纤维，使用电感耦合等离子体质谱法（ICP-MS）。我们的研究结果是：（1）Na，K-ATP酶的胞外底物位点与Rb+和K+结合的表观亲和力相当;然而，当Rb+是转运离子时，周转率降低;（2）Na，K-ATP酶对Rb+的摄取速率不是恒定的，而是随着约1.5 min的半衰期而下降;（3）α2同工酶对K+的表观K+亲和力显著低于α1。在存在10 µM哇巴因的情况下，在WT和α 1 S/Sα 2 R/R小鼠的完整纤维中进行测量时，α1和α2同工酶的K1/2、K分别为1.3和4 mM。总的来说，这些结果验证了单纤维模型的Na，K-ATP酶的运输和动力学常数的研究，他们意味着存在的机制，动态限制泵活性期间的主动运输。
The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K+ and Rb+ affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2−/−). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb+ and K+ with comparable apparent affinities; however; turnover rate is reduced when Rb+ is the transported ion; (2) The rate of Rb+ uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K+ affinity of the α2 isozymes for K+ is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes are 1.3 and 4 mM, respectively. Collectively, these results validate the single fiber model for studies of Na,K-ATPase transport and kinetic constants, and they imply the existence of mechanisms that dynamically limit pump activity during periods of active transport.
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