COMPARISON OF THE ENZYMATIC-PROPERTIES OF THE NA,K-ATPASE ALPHA-3-BETA-1 AND ALPHA-3-BETA-2 ISOZYMES
COMPARISON OF THE ENZYMATIC-PROPERTIES OF THE NA,K-ATPASE ALPHA-3-BETA-1 AND ALPHA-3-BETA-2 ISOZYMES
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DOI：
10.1021/bi00031a011
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发表时间：
1995-08-08
期刊：
影响因子：
2.9
通讯作者：
MERCER, RW
中科院分区：
文献类型：
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作者：
BLANCO, G;SANCHEZ, G;MERCER, RW
在哺乳动物组织中Na，K-ATP酶的α和β亚基的多种同种型的共表达引起了作为Na泵特征的复杂分子异质性。利用重组杆状病毒在昆虫细胞中表达不同的Na，K-ATP酶同工型代表了用于分析Na，K-ATP酶同工型功能的有用系统。在本研究中，我们使用该系统来指导大鼠Na，K-ATP酶α 3 β 1和α 3 β 2在Sf-9细胞中的表达，Sf-9细胞是来自秋粘虫Spodoptera frugiperda卵巢的细胞系。α 3与β 1或β 2的结合导致具有催化活性的Na，K-ATP酶同工酶。对这些酶的动力学特征的分析表明，伴随的β亚基同种型不会显著影响α 3多肽的性质。这是证明了类似的营业额数字，明显的亲和力K+和ATP，以及相当高的敏感性哇巴因表现出的两种同工酶。然而，两种同工酶对Na+的动力学依赖性不同，α 3 β 2对阳离子的表观亲和力比α 3 β 1高1.6倍。与其他Na，K-ATP酶同工酶的比较表明，α 3 β 2的表观Na+亲和力与在每个组织中广泛表达的α 1 β 1 Na泵相似;然而，其对K+、ATP和哇巴因的反应性是α 3同工酶的特征。Na，K-ATP酶功能中最显著的动力学差异是α亚型组成变化的结果。在这方面，最显著的动力学差异对应于对哇巴因的反应性，其中α 3 β 1和α 3 β 2显示出对强心类固醇的高敏感性，α 2 β 1和α 2 β 2显示出对强心类固醇的中等敏感性，而α 1 β 1显示出对强心类固醇的低敏感性。总的来说，对个别配体的酶的差异可能是重要的，在适应细胞的Na，K-ATP酶活性，以特定的生理要求。
The coexpression of multiple isoforms of the alpha and beta subunits of the Na,K-ATPase in mammalian tissues gives rise to the complex molecular heterogeneity that characterizes the Na pump. The expression of the different Na,K-ATPase isoforms in insect cells using recombinant baculoviruses represents a useful system for the analysis of Na,K-ATPase isoform function. In the present study, we use this system to direct the expression of the rat Na,K-ATPase alpha 3 beta 1 and alpha 3 beta 2 in Sf-9 cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. The association of alpha 3 with either beta 1 or beta 2 results in catalytically competent Na,K-ATPase isozymes. Analysis of the kinetic characteristics of these enzymes demonstrates that the accompanying beta subunit isoform does not drastically affect the properties of the alpha 3 polypeptide. This is evidenced by the similar turnover numbers, apparent affinities for K+ and ATP, and the comparable high sensitivity to ouabain exhibited by both isozymes. The kinetic dependence on Na+, however, is different for both isozymes, with alpha 3 beta 2 displaying a 1.6-fold higher apparent affinity for the cation than alpha 3 beta 1. Comparison with other Na,K-ATPase isozymes shows that the apparent Na+ affinity of alpha 3 beta 2 is similar to that of the alpha 1 beta 1 Na pump widely expressed in every tissue; nevertheless, its reactivity toward K+, ATP, and ouabain are characteristic of the alpha 3 isoform. The most pronounced kinetic differences in Na,K-ATPase function are a result of variations in alpha isoform composition. In this regard, the most conspicuous kinetic difference corresponds to the reactivity toward ouabain, with alpha 3 beta 1 and alpha 3 beta 2 displaying a high, alpha 2 beta 1 and alpha 2 beta 2 an intermediate, and alpha 1 beta 1 a low sensitivity for the cardiotonic steroid. Overall, the enzymatic differences toward the individual ligands may be important in adapting cellular Na,K-ATPase activity to specific physiological requirements.