Therien A G, Nestor N B, Ball W J, Blostein R
Department of Biochemistry, McGill University, Montreal, Canada.
J Biol Chem. 1996 Mar 22;271(12):7104-12. doi: 10.1074/jbc.271.12.7104.
The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na+ and K+ activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1-transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na+ of alpha1 pumps was axolemma approximately rat alpha1-transfected HeLa > kidney, and for K+, kidney approximately alpha1-transfected HeLa > axolemma. For alpha3, the order of apparent affinities for Na+ was pineal gland approximately axolemma > alpha3*-transfected HeLa, and for K+, alpha3*-transfected HeLa > axolemma approximately pineal gland. In addition, the differences in apparent affinities for Na+ of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K+ concentration was increased. A kinetic analysis of the apparent affinities for Na+ as a function of K+ concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na+ and K+, the latter acting as a competitive inhibitor at cytoplasmic Na+ activation sites. Although the nature of the tissue-specific modulation of K+/Na+ antagonism remains unknown, an analysis of the nature of the beta isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct beta subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances beta1 is the predominant beta isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K+/Na+ antagonism due to distinct betas may apply to alpha3 of axolemma (alpha3beta1) and pineal gland ( alpha3beta2).
本报告中描述的实验调和了早期研究中报道的钠钾ATP酶同工型特异性动力学方面一些明显的差异。因此，当在不同组织或细胞中检测同一同工型时，观察到同一物种（大鼠）钠钾ATP酶活性的钠和钾激活动力学存在组织特异性差异。以α1为例，比较了α1转染的HeLa细胞、大鼠肾脏和轴突膜。对于α3，比较了哇巴因不敏感的α3转染的HeLa细胞（参见Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925 - 16930）、松果体和轴突膜（主要是α3）。α1泵对钠的表观亲和力顺序为轴突膜≈大鼠α1转染的HeLa细胞＞肾脏，对钾的亲和力顺序为肾脏≈α1转染的HeLa细胞＞轴突膜。对于α3，对钠的表观亲和力顺序为松果体≈轴突膜＞α3转染的HeLa细胞，对钾的亲和力顺序为α3转染的HeLa细胞＞轴突膜≈松果体。此外，当钾浓度增加时，肾脏α1或HeLaα3与其他组织中同一同工型相比，对钠的表观亲和力差异更大。对钠的表观亲和力作为钾浓度函数的动力学分析表明，同工型特异性以及组织特异性差异与对钠和钾的表观亲和力有关，钾在细胞质钠激活位点起竞争性抑制剂的作用。尽管钾/钠拮抗作用的组织特异性调节的本质尚不清楚，但使用同工型特异性免疫沉淀分析与α1或α3相关的β同工型的性质表明，不同β亚基的存在并不能解释肾脏、轴突膜和HeLa细胞中α1的差异，以及轴突膜和HeLa细胞中α3的差异；在这两种情况下，β1都是存在的或与α1或α3相关的主要β同工型。然而，由于不同的β导致的钾/钠拮抗作用的动力学差异可能适用于轴突膜的α3（α3β1）和松果体的α3（α3β2）。
