Similarities and differences between the properties of native and recombinant Na+/K+-ATPases
Similarities and differences between the properties of native and recombinant Na+/K+-ATPases
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DOI：
10.1006/abbi.1996.0237
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发表时间：
1996-06-01
影响因子：
3.9
通讯作者：
Askari, A
中科院分区：
文献类型：
--
作者：
Xie, ZJ;Wang, YH;Askari, A
Na+/K+- atp酶的结构与其反应机制的关系的诱变研究进展受到目前可用表达系统中获得的少量不纯重组酶性质信息的缺乏以及在新环境中表达是否会改变该膜酶的各种催化活性的不确定性的阻碍，因此，我们的目的是详细比较广泛研究的犬肾Na+/K+- atp酶与杆状病毒感染的Sf-9细胞中表达的α (1), β(1)亚基的性质。重组酶的活性部分，含有10-20%表达的α亚基，被发现具有正常的摩尔活性，所有的部分反应，以及在重组为蛋白脂质体后催化atp依赖性Na+/K+交换的能力。重组酶和天然酶水解活性的稳态动力学比较表明：(a)两种制剂中Na+-ATP酶的ATP和Na+区是相同的；(b)重组酶对K+-磷酸酶的K+表观亲和力低于肾酶；(c)对于Na+/K+-ATP酶活性，重组酶的表观K+亲和力较低，其表观Na+和ATP亲和力高于肾酶。两种酶具有相似的ADP和K+敏感磷酸中间体，对瓦阿因的亲和力相同，对瓦阿因-酶复合物解离率的配体敏感性相似。显然，重组酶在细胞质K+位点的亲和力降低，但在多个Na+、ATP和瓦巴因结合位点没有变化，这种选择性变化的原因可能包括β糖基化状态的改变以及活性和非活性重组酶之间的相互作用。本研究结果为犬Na+/K+-ATPase α(1)、β(1)异构体的结构功能研究提供了必要的数据库，并表明需要对其他表达系统中获得的重组Na+/K+-ATPase进行类似的研究。(C) 1996学术出版社，Inc.
Progress of mutagenesis studies on the relation of the structure of Na+/K+-ATPase to its reaction mechanism has been impeded by the paucity of information on the properties of small amounts of impure recombinant enzyme obtained in the currently available expression systems, and the uncertainty of whether expression in a new environment alters the various catalytic activities of this membrane enzyme, Hence, our aim was to make a detailed comparison of the properties of the extensively studied canine kidney Na+/K+-ATPase with those of its alpha(1),beta(1) subunits expressed in the baculovirus-infected Sf-9 cells, The active fraction of the recombinant enzyme, containing 10-20% of the expressed alpha sub-units, was found to have normal molar activity, all the partial reactions, and the ability to catalyze ATP-dependent Na+/K+ exchange after reconstitution into proteoliposomes, Comparison of steady-state kinetics of the hydrolytic activities of recombinant and native enzymes showed that (a) ATP and Na+ plots of Na+-ATPase were the same in the two preparations; (b) apparent K+ affinity of K+-phosphatase of recombinant enzyme was lower than that of kidney enzyme; and (c) for Na+/K+-ATPase activity, apparent K+ affinity of recombinant enzyme was lower, and its apparent Na+ and ATP affinities were higher than those of kidney enzyme, The two enzymes had similar ADP- and K+-sensitive phosphointermediates, identical affinities for ouabain, and similar ligand sensitivities of dissociation rates of ouabain-enzyme complexes. Evidently, the recombinant enzyme has reduced affinity at cytoplasmic K+ sites, but no changes at multiple Na+, ATP, and ouabain binding sites, Likely causes of this selective change include altered glycosylation state of beta and interactions among active and inactive recombinant enzymes. The present results provide the necessary database for the appropriate use of an expression system in structure-function studies on canine alpha(1),beta(1) isoform of Na+/K+-ATPase, and indicate the need for similar studies on recombinant Na+/K+-ATPases obtained in other expression systems. (C) 1996 Academic Press, Inc.