Klodos I, Nørby J G, Plesner I W
Biochim Biophys Acta. 1981 May 6;643(2):463-82. doi: 10.1016/0005-2736(81)90089-4.
(1) The kinetics of the phosphorylated enzymic intermediates of (Na+ + K+)-ATPase from ox brain, which are formed by incubation of the enzyme with 25 microM AT32P, 150 mM Na+ and 1 mM Mg2+, have been studied in dephosphorylation experiments at 1 degree C. The dephosphorylation of the 32P-labelled enzyme was initiated by addition of either 1 mM unlabelled ATP, 2.5 mM ADP or 1 mM unlabelled ATP + ADP in concentrations from 25 to 1000 microM. (2) In the absence of ADP the dephosphorylation curve was linear in a semilogarithmic plot almost from t = 0, whereas by addition of ADP a biphasic behaviour was obtained. The slope of the slow phase of dephosphorylation was virtually independent of the ADP concentration. (3) The results were analysed by the mathematical equation corresponding to the simplest possible model for the interconversion and breakdown of the phosphointermediates: (formula: see text) where alpha, beta, H and G are functions of all the rate constants and H and G furthermore are functions of the initial values for [E1P] and [E2P]. (4) The analysis confirmed the model and enabled the determination of all the rate constants. (5) k-1 was found to be equal to k'-1 + k"-1 . [ADP] indicating an ADP-independent 'spontaneous' dephosphorylation of E1P. The rate constant for this process was close to that for dephosphorylation of E2P, i.e., k'-1 congruent to k3. Also the value of k"-1 was determined. (6) k3 was found to be at least 10 . k-2. The implication of this for the role of the E1P to E2P transition in the Na+ + K+)-stimulated ATP hydrolysis will be discussed in detail in the following paper (Plesner, I.W., Plesner, L., Nørby, J.G. and Klodos, I. (1981) Biochim. Biophys. Acta 643, 483--494). (7) A refinement of the model, accounting for the effect of Na+ on the steady-state ratio between [E1P] and [E2P] is proposed: (formula: see text). At [Na+] = 150 mM as used here, E1P(Na) and E'1P are assumed to be in rapid equilibrium. (8) Comparison of our results with those of others underlines the general validity of the conclusions of the present paper.
(1) 用来自牛脑的(Na⁺ + K⁺)-ATP酶与25微摩尔AT³²P、150毫摩尔Na⁺和1毫摩尔Mg²⁺一起温育形成的磷酸化酶中间体的动力学，已在1℃的去磷酸化实验中进行了研究。通过加入1毫摩尔未标记的ATP、2.5毫摩尔ADP或浓度为25至1000微摩尔的1毫摩尔未标记的ATP + ADP来启动32P标记酶的去磷酸化。(2) 在没有ADP的情况下，去磷酸化曲线在半对数图中几乎从t = 0开始就是线性的，而加入ADP后则呈现双相行为。去磷酸化慢相的斜率实际上与ADP浓度无关。(3) 结果通过与磷酸中间体的相互转化和分解的最简单可能模型相对应的数学方程进行分析：(公式：见原文)，其中α、β、H和G是所有速率常数的函数，而且H和G还是[E1P]和[E2P]初始值的函数。(4) 分析证实了该模型，并能够确定所有速率常数。(5) 发现k-1等于k'-1 + k"-1·[ADP]，这表明E1P存在不依赖于ADP的“自发”去磷酸化。该过程的速率常数接近于E2P去磷酸化的速率常数，即k'-1与k3相当。还确定了k"-1的值。(6) 发现k3至少是k-2的10倍。这对于E1P向E2P转变在(Na⁺ + K⁺)刺激的ATP水解中的作用的意义将在后续论文中详细讨论(Plesner, I.W., Plesner, L., Nørby, J.G.和Klodos, I. (1981) Biochim. Biophys. Acta 643, 483 - 494)。(7) 提出了该模型的一个改进版本，考虑了Na⁺对[E1P]和[E2P]之间稳态比率的影响：(公式：见原文)。在这里使用的[Na⁺] = 150毫摩尔时，假设E1P(Na)和E'1P处于快速平衡。(8) 将我们的结果与其他人的结果进行比较，强调了本文结论的普遍有效性。
