钠钾ATP酶的突变体Phe788→Leu被微摩尔浓度的钾抑制，并且在低钠浓度下表现出高钠ATP酶活性。

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

作者信息

Vilsen B

机构信息

Department of Physiology, University of Aarhus, Denmark.

出版信息

Biochemistry. 1999 Aug 31;38(35):11389-400. doi: 10.1021/bi990951t.

DOI:10.1021/bi990951t

PMID:10471289

Abstract

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).

摘要

将大鼠肾脏Na⁺,K⁺-ATP酶的Phe788突变为Leu在COS细胞中表达，活性位点浓度在40至60 pmol/mg膜蛋白之间。功能特性分析表明，该突变体严重损害了系统两侧对Na⁺和K⁺的区分能力。微摩尔浓度的K⁺抑制ATP水解（在20 mM Na⁺时，突变体的抑制半数抑制浓度（K(0.5)）为107 μM，而野生型为76 mM），在20 mM K⁺时，Na⁺,K⁺-ATP酶活性的分子周转数降至野生型的11%。这种抑制作用可被高浓度的Na⁺抵消，在完全没有K⁺的情况下，突变体以极高的速率催化Na⁺激活的ATP水解（“Na⁺-ATP酶活性”），相当于最大Na⁺,K⁺-ATP酶活性的86%。高Na⁺-ATP酶活性是由于K⁺非依赖性去磷酸化速率增加所致。在2 mM Na⁺时，突变体的去磷酸化速率就比野生型高8倍，Na⁺诱导的最大去磷酸化速率相当于K⁺诱导的去磷酸化速率的61%。K⁺对突变体中ATP水解产生抑制作用的原因是K⁺封闭的E2(K2)形式具有异常的稳定性。因此，当通过K⁺与未磷酸化的酶结合形成E2(K2)时，突变体中K⁺封闭的K(0.5)接近1 μM，而野生型为100 μM。在存在100 mM Na⁺以竞争K⁺结合的情况下，突变体中K⁺封闭的K(0.5)仍比野生型低100倍。此外，相对于野生型，突变体中封闭的K⁺释放速率降低了6 - 7倍，在对硝基苯磷酸酶（pNPPase）反应激活中表观K⁺亲和力增加了3 - 4倍，在存在250 μM K⁺的Na⁺,K⁺-ATP酶测定中表观ATP亲和力降低了10 - 11倍（K⁺-ATP拮抗作用增加），表观哇巴因亲和力降低了8倍（K⁺-哇巴因拮抗作用增加）。