AN UNEXPECTED EFFECT OF ATP ON THE RATIO BETWEEN ACTIVITY AND PHOSPHOENZYME LEVEL OF NA+/K+-ATPASE IN STEADY-STATE
AN UNEXPECTED EFFECT OF ATP ON THE RATIO BETWEEN ACTIVITY AND PHOSPHOENZYME LEVEL OF NA+/K+-ATPASE IN STEADY-STATE
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DOI：
10.1016/0005-2736(94)00229-i
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发表时间：
1995-01-26
影响因子：
3.4
通讯作者：
GARRAHAN, PJ
中科院分区：
文献类型：
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作者：
SCHWARZBAUM, PJ;KAUFMAN, SB;GARRAHAN, PJ
根据Albers-Post模型，Na+/K+-ATP酶催化的ATP水解需要至少顺序形成两个磷酸酶的构象(E(1)P和E(2)P)，然后是K+刺激的E(2)P水解。在本文中，我们表明，该模型是更一般的一类模型中的一个特殊情况，在所有这些模型中，稳态下atp酶活性（upsilon）与总磷酸酶水平（EP）之间的比率仅由磷酸构象之间的相互转化速率常数和去磷酸化速率常数决定。由于这些被认为不受ATP的影响，ATP酶活性和EP的底物曲线在形状上应该是相同的，因此upsilon/EP的比值应该与ATP的浓度无关。我们通过平行测量upsilon和EP作为[ATP]的函数，在没有或存在K+， Rb+或NH4+的非限制浓度的情况下验证了这一预测。在没有K+或其同族物的情况下，两条曲线都遵循Michaelis-Menten动力学，具有几乎相同的K- M值（0.16 μ M），因此upsilon/EP保持独立于[ATP]。在K+、Rb+或NH4+的存在下，upsilon和EP沿两个Michaelis-Menten方程的和随[ATP]的增加而增加。upsilon的双相反应是众所周知的，但据我们所知，我们的结果是第一次证明EP对[ATP]的反应也是双相的。在这些条件下，upsilon/EP比率随着[ATP]从19.8 s（-1）增加到40.1 s(-1)，沿双曲线在9.5 μ m处达到一半最大值。为了保持当前模型的有效性，似乎有必要假设ATP作用于E(1)P可逆箭头E(2)P转变和/或E(2)P的水解速率。后一种可能性被排除了。我们还发现，为了使阿尔伯斯-波斯特模型符合我们的数据，K+从E(2)中解封的速率常数必须比部分反应测量结果高10倍左右。结果表明，Albers-Post模型可以定量预测Na+- atp酶活性的实验行为，但不能定量预测Na+/K+- atp酶活性，除非包含其他尚未证实的假设。
According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K+-ATPase requires the sequential formation of at least two conformers of a phosphoenzyme (E(1)P and E(2)P), followed by the K+-stimulated hydrolysis of E(2)P. In this paper we show that this model is a particular case of a more general class of models in all of which the ratio between ATPase activity (upsilon) and total phosphoenzyme level (EP) in steady state is determined solely by the rate constants of interconversion between phosphoconformers and of dephosphorylation. Since these are thought to be unaffected by ATP, the substrate curves for ATPase activity and EP should be identical in shape so that the ratio upsilon/EP ought to be independent of the concentration of ATP. We tested this prediction by parallel measurements of upsilon and EP as a function of [ATP] in the absence or presence of non-limiting concentrations of K+, Rb+ or NH4+. In the absence of K+ or its congeners, both curves followed Michaelis-Menten kinetics, with almost identical K-m values (0.16 mu M) so that upsilon/EP remained independent of [ATP]. In the presence of either K+, Rb+ or NH4+, upsilon and EP increased with [ATP] along the sum of two Michaelis-Menten equations. The biphasic response of upsilon is well known but, to the best of our knowledge, our results are the first demonstration that the response of EP to [ATP] is also biphasic. Under these conditions, the ratio upsilon/EP increased with [ATP] from 19.8 to 40.1 s(-1) along a hyperbola that was half-maximal at 9.5 mu M. To preserve the validity of the current model it seems necessary to assume that ATP acts on the E(1)P reversible arrow E(2)P transition and/or on the rate of hydrolysis of E(2)P. The latter possibility was ruled out. We also found that to fit the Albers-Post model to our data, the rate constant for K+ deocclussion from E(2) has to be about 10-times higher than that reported from measurements of partial reactions. The results indicate that the Albers-Post model quantitatively predicts the experimental behavior of the Na+-ATPase ac tivity but is unable to do this for the Na+/K+-ATPase activity, unless additional and yet unproved hypothesis are included.