Hamer E, Schoner W
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, Germany.
Eur J Biochem. 1993 Apr 15;213(2):743-8. doi: 10.1111/j.1432-1033.1993.tb17815.x.
The chromium complex of adenosine 5'-[beta,gamma-methylene]triphosphate, Cr(H2O)4AdoPP[CH2]P, inactivates Na+/K(+)-ATPase from pig kidney at 37 degrees C with an inactivation velocity constant of 7.1 x 10(-3) min-1 by binding to the high-affinity ATP site (E1ATP site). The dissociation constant (Kd) of the analogue at this site is 26 microM, and of ATP 0.8 microM. Inactivation of the overall reaction of Na+/K(+)-ATPase by Cr(H2O)4AdoPP[CH2]P did not alter the activities of the E2 conformational state such as K(+)-activated p-nitrophenylphosphatase, 86Rb+ occlusion and [3H]ouabain binding by the 'backdoor' phosphorylation. However, [3H]ouabain binding via the forwards reaction from E1ATP in the presence of Na+ + Mg2+ is inhibited. K(+)-activated p-nitrophenylphosphatase activity of the Cr(H2O)4AdoPP[CH2]P-inactivated enzyme decreases when an MgATP analogue, the tetraammine cobalt complex of ATP, Co(NH3)4ATP, is used additionally to inactivate the E2ATP site. The enzyme activity of K(+)-activated phosphatase is also lost if the beta,gamma-bidentate chromium(III) complex of ATP, Cr(H2O)4ATP, which may form a stable E1-chromo-phosphointermediate, is used for the inactivation of Na+/K(+)-ATPase. We conclude that the phenomenon of a blockade of the overall reaction of Na+/K(+)-ATPase by the formation of a stable E1.CrAdoPP[CH2]P complex, leading thereby to a loss of the partial activities of the E1 conformation, but not of the E2 conformation, is consistent with the postulate of an (alpha beta)2 diprotomeric nature of the sodium pump. The observation, moreover, that treatment of the sodium pump with Cr(H2O)4ATP but not with Cr(H2O)4AdoPP[CH2]P leads to an inactivation of K(+)-activated phosphatase seems to indicate that the formation of a E1-phosphointermediate affects the E2ATP site.
腺苷 5'-[β,γ-亚甲基]三磷酸的铬配合物 Cr(H₂O)₄AdoPP[CH₂]P，在 37℃时通过与高亲和力 ATP 位点（E₁ATP 位点）结合，使猪肾中的 Na⁺/K⁺-ATP 酶失活，失活速度常数为 7.1×10⁻³ min⁻¹。该类似物在此位点的解离常数（Kd）为 26 μM，而 ATP 的解离常数为 0.8 μM。Cr(H₂O)₄AdoPP[CH₂]P 对 Na⁺/K⁺-ATP 酶总体反应的失活并未改变 E₂ 构象状态的活性，如 K⁺激活的对硝基苯磷酸酶、⁸⁶Rb⁺ 封闭以及通过“后门”磷酸化的[³H]哇巴因结合。然而，在 Na⁺ + Mg²⁺ 存在下，从 E₁ATP 通过正向反应的[³H]哇巴因结合受到抑制。当额外使用 MgATP 类似物 ATP 的四氨合钴配合物 Co(NH₃)₄ATP 使 E₂ATP 位点失活时，Cr(H₂O)₄AdoPP[CH₂]P 失活的酶的 K⁺激活的对硝基苯磷酸酶活性降低。如果使用可能形成稳定的 E₁-铬-磷酸中间体的 ATP 的 β,γ-双齿铬(III)配合物 Cr(H₂O)₄ATP 使 Na⁺/K⁺-ATP 酶失活，K⁺激活的磷酸酶的酶活性也会丧失。我们得出结论，通过形成稳定的 E₁·CrAdoPP[CH₂]P 配合物导致 Na⁺/K⁺-ATP 酶总体反应受阻，从而导致 E₁ 构象的部分活性丧失，但 E₂ 构象的部分活性未丧失，这一现象与钠泵的(αβ)₂ 二聚体性质的假设一致。此外，用 Cr(H₂O)₄ATP 而非 Cr(H₂O)₄AdoPP[CH₂]P 处理钠泵会导致 K⁺激活的磷酸酶失活，这一观察结果似乎表明 E₁-磷酸中间体的形成会影响 E₂ATP 位点。
