Grimaldi S, Pascale E, Pozzi D, D'Onofrio M, Giganti M G, Verna R
Istituto di Medicina Sperimentale C.N.R., Roma, Italy.
Biochim Biophys Acta. 1988 Sep 15;944(1):13-8. doi: 10.1016/0005-2736(88)90311-2.
The influence of occupancy by ouabain of its specific binding site on the stability and conformation of the Na+/K+-ATPase has been investigated. When native Na+/K+-ATPase is exposed to guanidinium chloride or diluted acid, tryptophanyl fluorescence falls to 50% of the initial value. If ouabain is bound, higher concentrations of GdmCl or acidity are needed to reach the same decrease in fluorescence. The rotational diffusion coefficient (relaxation time), shows higher values for the Na+/K+-ATPase (ouabain) complex compared to the enzyme alone, suggesting an increase in molecular asymmetry. This observation is confirmed by the Stern-Volmer analysis that shows an increase in the accessibility of the fluorophores in the Na+/K+-ATPase (ouabain) (KSV = 15.6 M-1) with respect to the native enzyme (KSV = 12.5 M-1). Iodine perturbation of the enzyme labelled with FITC, demonstrates a decrease in the accessibility of the fluorescein probe in the Na+/K+-ATPase(ouabain) (KSV = 4 M-1) compared to the Na+/K+-ATPase (KSV = 7 M-1) indicating that after ouabain binding this site of the enzyme is less exposed to the solvent. These data, in agreement with other reports, suggest an allosteric effect of ouabain binding on the Na+/K+-ATPase conformation.
哇巴因占据其特异性结合位点对Na⁺/K⁺-ATP酶稳定性和构象的影响已得到研究。当天然Na⁺/K⁺-ATP酶暴露于氯化胍或稀酸时，色氨酸荧光降至初始值的50%。如果结合了哇巴因，则需要更高浓度的GdmCl或酸度才能使荧光产生相同程度的降低。与单独的酶相比，Na⁺/K⁺-ATP酶（哇巴因）复合物的旋转扩散系数（弛豫时间）显示出更高的值，这表明分子不对称性增加。Stern-Volmer分析证实了这一观察结果，该分析表明，相对于天然酶（KSV = 12.5 M⁻¹），Na⁺/K⁺-ATP酶（哇巴因）中荧光团的可及性增加（KSV = 15.6 M⁻¹）。用FITC标记的酶的碘扰动表明，与Na⁺/K⁺-ATP酶（KSV = 7 M⁻¹）相比，Na⁺/K⁺-ATP酶（哇巴因）中荧光素探针的可及性降低（KSV = 4 M⁻¹），这表明哇巴因结合后酶的该位点较少暴露于溶剂中。这些数据与其他报告一致，表明哇巴因结合对Na⁺/K⁺-ATP酶构象具有变构效应。
