Blanco G, Sánchez G, Mercer R W
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1995 Aug 8;34(31):9897-903. doi: 10.1021/bi00031a011.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
钠钾ATP酶α和β亚基的多种同工型在哺乳动物组织中的共表达产生了构成钠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钠钾ATP酶同工型，是分析钠钾ATP酶同工型功能的有用系统。在本研究中，我们利用该系统在sf-9细胞（一种源自草地贪夜蛾卵巢的细胞系）中指导大鼠钠钾ATP酶α3β1和α3β2的表达。α3与β1或β2的结合产生具有催化活性的钠钾ATP酶同工酶。对这些酶的动力学特征分析表明，与之相伴的β亚基同工型不会显著影响α3多肽的特性。这两种同工酶的周转数、对K+和ATP的表观亲和力以及对哇巴因的相当高的敏感性都证明了这一点。然而，两种同工酶对Na+的动力学依赖性不同，α3β2对该阳离子的表观亲和力比α3β1高1.6倍。与其他钠钾ATP酶同工酶比较表明，α3β2的表观Na+亲和力与在每个组织中广泛表达的α1β1钠泵相似；然而，它对K+、ATP和哇巴因的反应性是α3同工型的特征。钠钾ATP酶功能最显著的动力学差异是α同工型组成变化的结果。（摘要截短至250字）
