COMPARISON OF THE EFFECTS OF POTASSIUM ON OUABAIN BINDING TO NATIVE AND SITE-DIRECTED MUTANTS OF NA,K-ATPASE
COMPARISON OF THE EFFECTS OF POTASSIUM ON OUABAIN BINDING TO NATIVE AND SITE-DIRECTED MUTANTS OF NA,K-ATPASE
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DOI：
10.1006/abbi.1995.1145
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发表时间：
1995-02-20
影响因子：
3.9
通讯作者：
WALLICK, ET
中科院分区：
文献类型：
--
作者：
JOHNSON, CL;SCHULTHEIS, PJ;WALLICK, ET
我们检测了K+对Mg 2（+）-和P-i-支持的[H-3]哇巴因与Na，K-ATP酶结合的影响，所述Na，K-ATP酶包括来自绵羊肾脏的部分纯化的酶以及在NIH 3 T3细胞中表达的野生型和突变型绵羊α 1亚型（C104 A，Y108 A，E116 Q，P118 K，Y124 F，R880 P，R880 L和N122 D）。在K+浓度增加的情况下，[H-3]哇巴因与这些酶的结合减少，但从未达到非特异性结合水平，这与哇巴因仍然能够与K+复合酶结合但亲和力降低的概念一致。K+抑制哇巴因结合的部分竞争模型，提出了令人满意的拟合的结合数据。该模型与两个K+离子与酶的顺序结合是一致的。从该模型获得的K+的Ki值（约1.0 mM）与通过测量Na，K-ATP酶活性的K+依赖性测定的大鼠α亚型的表观K+亲和力相当[E. A.朱厄尔和J. B. Lingrel（1991）J.Biol.Chem.266，16925-16930]。这与Mg 2+加Pi支持的哇巴因结合的K+抑制是由K+结合到生理条件下天然酶中存在的相同高亲和力结合位点介导的概念一致。虽然突变体表现出哇巴因的结合常数，从野生型酶的变化超过30倍，其对K+的亲和力从天然酶的差异小于两倍。我们的研究结果表明，哇巴因和K+结合位点是不相同的，差异的酶的突变的影响。这里描述的系统应证明是有用的阳离子结合的Na，K-ATP酶的其他突变体的分析，特别是那些携带氨基酸取代，导致一个失活的酶。(C)出版社：Academic Press
We examined the effect of K+ on Mg2(+)- and P-i-supported [H-3] ouabain binding to Na,K-ATPases, including partially purified enzyme from sheep kidney and wild-type and mutant sheep alpha 1 isoforms (C104A, Y108A, E116Q, P118K, Y124F, R880P, R880L, and N122D) expressed in NIH3T3 cells. In the presence of increasing concentrations of K+, [H-3] ouabain binding to these enzymes decreases but never reaches nonspecific binding levels, consistent with the concept that ouabain is still able to bind to the K+-complexed enzyme but with reduced affinity. A partially competitive model for K+ inhibition of ouabain binding is proposed which satisfactorily fits the binding data. The model is consistent with the sequential binding of two K+ ions to the enzyme. K-i values (approximate to 1.0 mM) for K+ obtained from this model are comparable to the apparent K+ affinities of the rat alpha isoforms determined by measuring the K+ dependence of Na,K-ATPase activity [E. A. Jewell and J. B. Lingrel (1991) J. Biol. Chem. 266, 16925-16930]. This is consistent with the concept that K+ inhibition of Mg2+ plus P-i supported ouabain binding is mediated by K+ binding to the same high-affinity binding sites present in the native enzyme under physiological conditions. While the mutants exhibit binding constants for ouabain which vary more than 30-fold from that of the wild-type enzyme, their affinities for K+ differ less than twofold from that of the native enzyme. Our results suggest that the ouabain and K+ binding sites are not the same and are differentially affected by mutations of the enzyme. The system described here should prove useful in the analysis of cation binding to other mutants of the Na,K-ATPase, especially those carrying amino acid replacements which result in an inactive enzyme. (C) 1995 Academic Press, Inc.