（钠+ +钾+）-ATP酶：钠-ATP酶活性磷酸中间产物三池模型的确认。酶-ATP解离速率常数的估算。

(Na+ + K+)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na+-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant.

作者信息

Klodos I, Nørby J G

出版信息

Biochim Biophys Acta. 1987 Feb 26;897(2):302-14. doi: 10.1016/0005-2736(87)90426-3.

Abstract

The dephosphorylation kinetics of acid-stable phosphointermediates of (Na+ + K+)-ATPase from ox brain, ox kidney and pig kidney was studied at 0 degree C. Experiments performed on brain enzyme phosphorylated at 0 degree C in the presence of 20-600 mM Na+, 1 mM Mg2+ and 25 microM [gamma-32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na+ concentration, all phosphoenzyme is either ADP- or K+-sensitive. After phosphorylation of kidney enzymes at 0 degree C with 1 mM Mg2+, 25 microM [gamma-32P]ATP and 150-1000 mM Na+ the amounts of ADP- and K+-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.5 mM ADP or 1 mM ATP + 20 mM K+. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na+, after addition of 1 mM ATP + 20 mM K+ is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E1 approximately P(Na3) as intermediate of (Na+ + K+)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K+-sensitive phosphoenzymes is greater than E tot. In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k-1, is obtained. k-1 varies between 1 and 4 s-1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na+ or Tris+. The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na+-ATPase activity.

摘要

在0℃下研究了来自牛脑、牛肾和猪肾的（Na⁺+K⁺）-ATP酶的酸稳定磷酸中间产物的去磷酸化动力学。在0℃下，于20 - 600 mM Na⁺、1 mM Mg²⁺和25 μM [γ-³²P]ATP存在下对脑酶进行磷酸化实验，结果表明，无论由Na⁺浓度决定的EP池组成如何，所有磷酸酶对ADP或K⁺均敏感。在0℃下，用1 mM Mg²⁺、25 μM [γ-³²P]ATP和150 - 1000 mM Na⁺对肾酶进行磷酸化后，通过添加1 mM ATP + 2.5 mM ADP或1 mM ATP + 20 mM K⁺来测定对ADP和K⁺敏感的磷酸酶的量。与先前关于脑酶的报道结果相似，两种类型的去磷酸化曲线均有一个快速相和一个慢速相，因此在添加1 mM ATP + 20 mM K⁺后，对于肾酶也观察到一部分磷酸酶缓慢衰减，在1000 mM Na⁺时高达80%。因此，用肾酶获得的结果似乎强化了先前对E1≈P(Na3)作为（Na⁺+K⁺）-ATP酶活性中间体所起作用的怀疑。此外，对于两种肾酶，对ADP和K⁺敏感的磷酸酶之和大于E总量。在对脑酶的实验中，获得了酶-ATP复合物的解离速率常数k-1的估计值。k-1在1至4 s⁻¹之间变化，似乎取决于复合物形成过程中存在的配体。在Na⁺或Tris⁺存在下形成的酶-ATP复合物的值最高。结果证实了三池模型在描述Na⁺-ATP酶活性的磷酸中间产物的去磷酸化动力学方面的有效性。