Koepsell H
J Membr Biol. 1978 Dec 8;44(1):85-102. doi: 10.1007/BF01940575.
Antibodies which were raised against highly purified membrane-bound (Na+ -K+)-ATPase from the outer medulla of rat kidneys inhibit the (Na+-K+)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots. In the purified membrane-bound (Na+-K+)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na+ -K+)-ATPase activity (n = 0.86), (b) for the ATP binding to the enzyme (n = 0.58), and (c) for the ouabain inhibition of the (Na+ -K+)-ATPase activity (n = 0.77). By measuring the Na+ dependence of the (Na+ -K+)-ATPase reaction, a positive homotropic cooperativity (n = 1.67) was found. As reactivation of the antibody-inhibited enzyme proceeds very slowly (t0.5 = 5.2 hr), it was possible to measure characteristics of the antibody-(Na+ -K+)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na+ -K+)-ATPase reaction and on the ATP binding of the enzyme. Vmax of the (Na+ -K+)-ATPase reaction and the number of ATP binding sites were reduced while K0.5 ATP for the (Na+ -K+)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased the K0.5 value for the Na+ stimulation of the (Na+ -K+)-ATPase activity, but they did not alter the homotropic interactions between the Na+-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na+ -K)-ATPase activity was abolished by the antibodies. The data are tentatively explained by the following model: The antibodies bind to the (Na+ -K+)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na+ -K+)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na+ dependence of the (Na+ -K+)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.
针对从大鼠肾脏外髓质高度纯化的膜结合（Na⁺ - K⁺）-ATP酶产生的抗体，可将（Na⁺ - K⁺）-ATP酶活性抑制高达95%。抗体抑制是可逆的。在半对数图中，酶抑制和重新激活的时间进程呈双相性。在纯化的膜结合（Na⁺ - K⁺）-ATP酶中，观察到了负协同性：（a）对于（Na⁺ - K⁺）-ATP酶活性的ATP依赖性（n = 0.86）；（b）对于ATP与该酶的结合（n = 0.58）；（c）对于哇巴因对（Na⁺ - K⁺）-ATP酶活性的抑制作用（n = 0.77）。通过测量（Na⁺ - K⁺）-ATP酶反应的Na⁺依赖性，发现了正同促协同性（n = 1.67）。由于抗体抑制的酶重新激活过程非常缓慢（t0.5 = 5.2小时），因此有可能测量抗体 - （Na⁺ - K⁺）-ATP酶复合物的特性：抗体对（Na⁺ - K⁺）-ATP酶反应的ATP依赖性以及酶的ATP结合产生类似影响。（Na⁺ - K⁺）-ATP酶反应的Vmax和ATP结合位点数量减少，而抗体使（Na⁺ - K⁺）-ATP酶活性和ATP结合的K0.5 ATP增加。抗体对ATP结合以及酶活性的ATP依赖性的希尔系数没有显著改变。抗体增加了（Na⁺ - K⁺）-ATP酶活性受Na⁺刺激的K0.5值，但未改变Na⁺结合位点之间的同促相互作用。抗体消除了观察到的哇巴因对（Na⁺ - K）-ATP酶活性抑制的负协同性。这些数据初步由以下模型解释：抗体从内膜侧与（Na⁺ - K⁺）-ATP酶结合，在ATP结合位点对称地减少ATP结合，从而也降低了该酶的（Na⁺ - K⁺）-ATP酶活性。抗体可能通过直接相互作用或通过ATP结合位点的构象变化来抑制ATP结合。这可能还导致了（Na⁺ - K⁺）-ATP酶活性的Na⁺依赖性改变以及观察到的对哇巴因抑制的剂量反应改变。
