Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.
Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.
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DOI：
10.1085/jgp.82.6.725
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发表时间：
1983-12
期刊：
影响因子：
--
通讯作者：
Christiansen NO
中科院分区：
文献类型：
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作者：
Nørby JG;Klodos I;Christiansen NO
为了确定Na+转运的生化事件，我们研究了Na+， Tris+和K+与牛脑中Na，K- atp酶磷酸化中间体的相互作用。在0℃下，用1 mM Mg2+、25微米[32P]ATP和20-600 mM Na+（含或不含Tris+）对该酶进行磷酸化，并研究了[32P]EP在加入(1)1 mM ATP、(2)2.5 mM ADP、(3)1 mM ATP加20 mM K+和(4)2.5 mM ADP加Na+至600 mM后的去磷酸化动力学。在去磷酸化类型2-4中，曲线为双相或多相。“adp敏感电位”和“K+敏感电位”是由曲线的慢相向纵坐标外推确定的，它们的和总是大于Etotal。这些结果需要一个最小模型，包括三个连续的EP池，a， B和C，其中a是ADP敏感的，B和C都是K+敏感的。在高[Na+]时，B迅速转化为A（第4型实验）。七个速率系数依赖于[Na+]， [Tris+]和[K+]，为了解释这一点，我们开发了一个阳离子与EP相互作用的综合模型。该模型具有以下特征：A、B、C为EP形态的平衡混合物；EP中的A有2 - 3个Na离子结合在高亲和力（内部）位点，池B有3个，池C有2 - 3个低亲和力（外部）位点。推测的Na+位点外的高亲和力可能在c池的E2P上。A导致B的转化被K+（和Tris+）阻断。我们得出结论，池A只能是Na- atp酶反应的中间产物，而不是Na，K泵的正常运行的中间产物。
To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. "ADP-sensitive EP" and "K+-sensitive EP" were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of three consecutive EP pools, A, B, and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The seven rate coefficients were dependent on [Na+], [Tris+], and [K+], and to explain this we developed a comprehensive model for cation interaction with EP. The model has the following features: A, B, and C are equilibrium mixtures of EP forms; EP in A has two to three Na ions bound at high-affinity (internal) sites, pool B has three, and pool C has two to three low-affinity (external) sites. The putative high- affinity outside Na+ site may be on E2P in pool C. The A leads to B conversion is blocked by K+ (and Tris+). We conclude that pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.