Taniguchi K, Suzuki K, Sasaki T, Shimokobe H, Iida S
J Biochem. 1986 Nov;100(5):1231-9. doi: 10.1093/oxfordjournals.jbchem.a121828.
Addition of up to 300 microM ATP in the presence of 2 M NaCl with MgCl2 to pig kidney Na+,K+-ATPase treated with N-[p-(2-benzimidazolyl)phenyl]maleimide seemed to be insufficient to saturate the rate of the fluorescence decrease. However, both the extent of the decrease and the amount of phosphoenzyme at a steady state were saturated below 20 microM ATP. Addition of Mg2+ with Na+ to the enzyme preincubated with 20 to 600 microM ATP gave nearly the same rate constant, which was below 50% of that obtained by adding 300 microM ATP to the Na+-form enzyme in the presence of Mg2+. High concentrations of ATP affected neither the rate of light-scattering change (Taniguchi, K. et al. (1986) J. Biol. Chem. 261, 3272-3281) after ADP-sensitive phosphoenzyme formation (E1P) nor that of the breakdown of E1P. A stoichiometric amount of [32P]Pi was liberated from [32P]E1P. The data suggested that ATP did not bind to E1P in such a way as to increase the extent of phosphorylation further or to accelerate dephosphorylation. The data also suggested that the reason for the large difference in the apparent affinity of ATP as evaluated from the rate and the extent of fluorescence change is the large dissociation constant for ATP of a Michaelis complex.
在含有2 M NaCl和MgCl₂的条件下，向用N-[对-(2-苯并咪唑基)苯基]马来酰亚胺处理过的猪肾Na⁺,K⁺-ATP酶中添加高达300 μM的ATP，似乎不足以使荧光降低速率达到饱和。然而，在20 μM ATP以下，荧光降低程度和稳态下磷酸酶的量均达到饱和。向预先用20至600 μM ATP孵育的酶中添加Mg²⁺和Na⁺，得到的速率常数几乎相同，该速率常数低于在Mg²⁺存在下向Na⁺型酶中添加300 μM ATP所获得速率常数的50%。高浓度的ATP既不影响ADP敏感的磷酸酶（E1P）形成后光散射变化的速率（Taniguchi, K.等人（1986年）《生物化学杂志》261, 3272 - 3281），也不影响E1P的分解速率。化学计量的[³²P]Pi从[³²P]E1P中释放出来。数据表明，ATP与E1P结合的方式不会进一步增加磷酸化程度或加速去磷酸化。数据还表明，从荧光变化速率和程度评估的ATP表观亲和力存在巨大差异的原因是米氏复合物中ATP的解离常数很大。
