Taniguchi K, Suzuki K, Iida S
J Biol Chem. 1982 Sep 25;257(18):10659-67.
The addition of Mg2+ or ATP to (Na+,K+)-ATPase (EC 3.6.1.3) of pig kidney modified with a sulfhydryl fluorescent reagent N-[p-(2-benzimidazolyl)phenyl]maleimide simply reduced fluorescence in the presence of Na+; however, the addition of both ligands to the enzyme induced a reversible dynamic change. The direction of the change was dependent on the concentration of Na+ present. These dynamic changes in fluorescence intensity both in the presence of low and high concentrations of Na+ can be repeated by the re-addition of ATP but not by ADP. Addition of ouabain under the former condition stabilized the fluorescence at the highest level, but the addition of ouabain under the latter condition increased the fluorescence from the lowest to the highest level. The phosphoenzyme formed under the former condition was sensitive to K+ and insensitive to ADP while the phosphoenzyme formed under the latter condition was sensitive to ADP and insensitive to K+. The data indicate that the positive and negative fluorescence changes were induced by the formation of K+-sensitive phosphoenzyme and ADP-sensitive phosphoenzyme, respectively. N-Ethylmaleimide treatment partially inhibited the positive change without affecting the negative change. These data also indicate that the transition of ADP-sensitive phosphoenzyme to K+-sensitive phosphoenzyme accompanied the largest fluorescence intensity change which was examined during the hydrolysis of ATP. The data obtained from the tryptophan fluorescence of both the native and the modified enzyme suggest that the micro-environments of the tryptophan and the sulfhydryl residues are similar in the state of K+-sensitive phosphoenzyme but different in the state of ADP-sensitive phosphoenzyme.
用巯基荧光试剂N-[对-(2-苯并咪唑基)苯基]马来酰亚胺修饰猪肾的(Na⁺,K⁺)-ATP酶（EC 3.6.1.3）后，添加Mg²⁺或ATP在有Na⁺存在时只会简单地降低荧光；然而，向该酶中同时添加这两种配体则会诱导可逆的动态变化。变化的方向取决于存在的Na⁺浓度。在低浓度和高浓度Na⁺存在下荧光强度的这些动态变化都可以通过重新添加ATP来重复，但不能通过添加ADP来重复。在前一种条件下添加哇巴因会使荧光稳定在最高水平，但在后一种条件下添加哇巴因会使荧光从最低水平增加到最高水平。在前一种条件下形成的磷酸酶对K⁺敏感而对ADP不敏感，而后一种条件下形成的磷酸酶对ADP敏感而对K⁺不敏感。数据表明，正向和负向荧光变化分别是由K⁺敏感的磷酸酶和ADP敏感的磷酸酶的形成所诱导的。N-乙基马来酰亚胺处理部分抑制了正向变化而不影响负向变化。这些数据还表明，ADP敏感的磷酸酶向K⁺敏感的磷酸酶的转变伴随着在ATP水解过程中检测到的最大荧光强度变化。从天然酶和修饰酶的色氨酸荧光获得的数据表明，色氨酸和巯基残基的微环境在K⁺敏感的磷酸酶状态下相似，但在ADP敏感的磷酸酶状态下不同。
