Boldyrev A A, Rubtsov A M, Lopina O D, McStay D, Yang L, Quinn P J
School of Applied Sciences, Robert Gordon University, Aberdeen, Scotland, UK.
Biokhimiia. 1995 Jul;60(7):1171-8.
Phosphorescence anisotropy of eosin-5'-isothiocyanate labelled Na,K-ATPase purified from duck salt glands has been studied. The initial anisotropy value is 0.235 +/- 0.015 (room temperature) and does not depend on the enzyme conformation (sodium or potassium). The experimental curve is fitted into a two-exponential curve with residual term, the fast component corresponds to the rotational mobility of the functional unit of Na,K-ATPase (promoter), while the slow one--to that of larger associates. In the presence of ligands modifying the conformational state of Na,K-ATPase (sodium, potassium, ATP) the rotational mobility of the fast component does not change in contrast with the slow one. A comparison of the enzyme rotational mobility in the presence of ligands simulating different steps of hydrolytic cycle suggests that interprotomer interactions are changed in the course of hydrolytic cycle: the fraction of larger associates increases at the step of the enzyme interactions with potassium ions, whereas their mobility in the bilayer enhances sharply after interaction with ATP. In the presence of the 2% non-ionic detergent, C12E9, the initial anisotropy value decreases down to 0.1; the residual term disappears thereby, while the curve is still two-exponential. However, the difference in the rotational mobility of sodium and potassium conformers diminishes. At the same time, the ratios between protomers and oligomers in the presence of sodium and potassium become approximated. This indicates that in the presence of the detergent high molecular weight associates are solubilized, the mobility of the both protomers and oligomers of Na,K-ATPase increases, while the difference between the mobilities of sodium and potassium conformers is disappeared.
对从鸭盐腺中纯化得到的异硫氰酸荧光素-5'-标记的钠钾-ATP酶的磷光各向异性进行了研究。初始各向异性值为0.235±0.015（室温），且不依赖于酶的构象（钠或钾）。实验曲线拟合为带有残差项的双指数曲线，快速成分对应于钠钾-ATP酶功能单元（启动子）的旋转流动性，而慢速成分对应于较大聚集体的旋转流动性。在存在改变钠钾-ATP酶构象状态的配体（钠、钾、ATP）时，快速成分的旋转流动性与慢速成分相反，没有变化。对模拟水解循环不同步骤的配体存在时酶的旋转流动性进行比较表明，在水解循环过程中，原聚体间相互作用发生了变化：在酶与钾离子相互作用的步骤中，较大聚集体的比例增加，而它们在双层膜中的流动性在与ATP相互作用后急剧增强。在存在2%的非离子去污剂C12E9时，初始各向异性值降至0.1；残差项由此消失，而曲线仍为双指数曲线。然而，钠和钾构象体旋转流动性的差异减小。同时，在存在钠和钾时原聚体和寡聚体之间的比例变得接近。这表明在去污剂存在下，高分子量聚集体被溶解，钠钾-ATP酶的原聚体和寡聚体的流动性均增加，而钠和钾构象体流动性之间的差异消失。
