Matsumoto A, Morita F
J Biochem. 1983 Apr;93(4):943-53. doi: 10.1093/oxfordjournals.jbchem.a134249.
Tyr 180 of chicken breast muscle alkali light chain A1 was nitrated with tetranitromethane. The nitroA1 was incorporated into chicken breast muscle subfragment-1 (S-1) by exchange with the intrinsic alkali light chain. In the presence of adenylylimidodiphosphate (AMPPNP) or ADP, the S-1 containing nitroA1 showed a difference visible absorption spectrum by Mg2+ or Ca2+. The difference spectrum has a trough around 435 nm, indicating a blue shift of the absorption spectrum due to the nitrophenol chromophore of the modified A1. The plot of delta A at 435 nm versus concentration of free Mg2+ fitted a single binding curve, independent of the total concentration of AMPPNP. These results reveal that free Mg2+ binds to the active site of S-1 ATPase, but not as Mg-AMPPNP complex. The dissociation constants of magnesium from S-1 complex were different with the two nucleotides and were 1.25 X 10(-8) M and 1.24 X 10(-7) with AMPPNP and ADP, respectively. The difference spectrum was also obtained in the presence of ATP. The delta epsilon value after adding ATP changed with the ATPase reaction. The steady state rate of S-1 ATPase was measured at various concentrations of free Mg2+. The dissociation constant of magnesium from the steady state complex, EPADP(a), was estimated as 6 X 10(-8) M. These results suggest that the affinity of magnesium at the active site of ATPase changes with the intermediate states of ATPase reaction. The affinity of calcium was lower than that of magnesium.
鸡胸肌碱性轻链A1的酪氨酸180被四硝基甲烷硝化。通过与内在碱性轻链交换，将硝基A1掺入鸡胸肌亚片段-1（S-1）中。在腺苷酰亚胺二磷酸（AMPPNP）或ADP存在下，含有硝基A1的S-1在Mg2+或Ca2+存在下呈现出可见吸收差异光谱。该差异光谱在435nm左右有一个低谷，表明由于修饰后的A1的硝基苯酚发色团导致吸收光谱发生蓝移。435nm处的ΔA与游离Mg2+浓度的关系图符合单一结合曲线，与AMPPNP的总浓度无关。这些结果表明，游离Mg2+与S-1 ATP酶的活性位点结合，但不是以Mg-AMPPNP复合物的形式。Mg2+从S-1复合物中的解离常数在两种核苷酸存在下不同，与AMPPNP和ADP结合时分别为1.25×10^(-8)M和1.24×10^(-7)M。在ATP存在下也获得了差异光谱。加入ATP后的Δε值随ATP酶反应而变化。在不同游离Mg2+浓度下测量了S-1 ATP酶的稳态速率。从稳态复合物EPADP(a)中Mg2+的解离常数估计为6×10^(-8)M。这些结果表明，ATP酶活性位点处Mg2+的亲和力随ATP酶反应的中间状态而变化。Ca2+的亲和力低于Mg2+。
