Peluffo R D, Rossi R C, Garrahan P J, Rega A F
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
J Biol Chem. 1994 Jan 14;269(2):1051-6.
The maximum rate of phosphorylation (rm) of a highly purified Na,K-ATPase from red outer medulla of pig kidney was measured at 25 degrees C as a function of ATP concentration in media with Mg2+, Na+, and no K+. When rm was plotted as a function of the concentration of ATP a biphasic response was observed with a hyperbolic component of high affinity (Km = 15.7 +/- 2.6 microM) and low velocity ((rm)max = 460 +/- 40 nmol of Pi/(mg of protein.s)) plus a parabolic component which showed no saturation up to 1000 microM ATP, concentration at which rm was 1768.1 +/- 429.6 nmol Pi/(mg protein.s) (mean +/- S.E.; n = 3). This low affinity effect of ATP on the rate of phosphorylation disappeared when the Na,K-ATPase underwent turnover in medium without K+ suggesting that, like superphosphorylation (Peluffo, R. D., Garrahan, P. J., and Rega, A. F. (1992) J. Biol. Chem. 267, 6596-6601), it required the enzyme to be at rest. This property of the Na,K-ATPase was not predicted by the Albers-Post reaction scheme. The observed behavior of the enzyme could be simulated by a scheme that involves a resting enzyme (Er) functionally different from E1 or E2, which is able to bind three molecules of ATP, one with high and two with low affinity, and that after phosphorylation is converted into the phosphointermediates that are generally considered to participate in the reaction cycle described by Albers and Post.
在25摄氏度下，测定了来自猪肾外髓质的高度纯化的钠钾ATP酶在含有镁离子、钠离子且无钾离子的培养基中磷酸化的最大速率（rm），该速率是ATP浓度的函数。当将rm绘制为ATP浓度的函数时，观察到双相反应，其中有一个高亲和力的双曲线成分（Km = 15.7 +/- 2.6微摩尔）和低速度（（rm）max = 460 +/- 40纳摩尔无机磷/（毫克蛋白质·秒）），再加上一个抛物线成分，该成分在高达1000微摩尔ATP时未显示饱和，此时rm为1768.1 +/- 429.6纳摩尔无机磷/（毫克蛋白质·秒）（平均值 +/- 标准误；n = 3）。当钠钾ATP酶在无钾离子的培养基中周转时，ATP对磷酸化速率的这种低亲和力效应消失，这表明，与超磷酸化一样（佩卢福，R. D.，加拉汉，P. J.，和雷加，A. F.（1992年）《生物化学杂志》267，6596 - 6601），它需要酶处于静止状态。钠钾ATP酶的这一特性无法由阿尔伯斯 - 波斯特反应模式预测。该酶观察到的行为可以通过一个模式来模拟，该模式涉及一种功能上不同于E1或E2的静止酶（Er），它能够结合三个ATP分子，一个具有高亲和力，两个具有低亲和力，并且在磷酸化后转化为通常被认为参与阿尔伯斯和波斯特描述的反应循环的磷酸中间产物。
