FUNCTIONAL EXPRESSION OF THE ALPHA-2-ISOFORMS AND ALPHA-3-ISOFORMS OF THE NA,K-ATPASE IN BACULOVIRUS-INFECTED INSECT CELLS
FUNCTIONAL EXPRESSION OF THE ALPHA-2-ISOFORMS AND ALPHA-3-ISOFORMS OF THE NA,K-ATPASE IN BACULOVIRUS-INFECTED INSECT CELLS
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DOI：
10.1073/pnas.90.5.1824
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发表时间：
1993-03-01
影响因子：
11.1
通讯作者：
MERCER, RW
中科院分区：
文献类型：
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作者：
BLANCO, G;XIE, ZJ;MERCER, RW
Na，K-ATP酶的α和β亚基的多种同种型已被鉴定。由于大多数组织表达多种同种型，并且尚未实现对每种同种型特异性的纯化技术，因此阐明它们的作用变得复杂。利用苜蓿银纹夜蛾杆状病毒（Autographica californica）感染昆虫细胞的杆状病毒表达系统是研究Na，K-ATP酶同工型的理想系统，因为它可以产生大量的外源蛋白，而某些昆虫细胞系具有低水平的内源Na，K-ATP酶。制备了含有大鼠Na，K-ATP酶α 2、α 3和β 1亚型cDNA的重组杆状病毒，并将其用于感染Sf-9细胞，Sf-9细胞是一种来源于秋粘虫草地贪夜蛾卵巢的昆虫细胞系。通过使用该系统，产生了在抗原性和免疫学上与天然亚基不可区分的Na，K-ATP酶α 2和α 3亚基。当每个亚基在Sf-9细胞中独立表达时，它主要被递送到质膜。虽然每个Na，K-ATP酶亚基的单独表达不能使Na，K-ATP酶分子具有活性，但α 2或α 3与β 1的共表达产生催化活性分子。这种活性可以作为哇巴因敏感的ATP酶活性来测量，或者使用[γ-P-32]ATP或P-32 i来直接证明，以鉴定α 2和α 3亚型的磷酸化中间体。[H-3]哇巴因结合研究表明，两种亚型都能够以高亲和力结合强心类固醇，α 3对哇巴因更敏感。这些结果表明，杆状病毒系统是合适的表达的Na，K-ATP酶的异构体，并应提供一个有用的方法，每个异构体的酶学性质的表征。
Multiple isoforms of both the alpha and beta subunits of Na,K-ATPase have been identified. Elucidating their roles has been complicated by the fact that most tissues express multiple isoforms and purification techniques specific for each isoform have not been achieved. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, is an ideal system for studying the Na,K-ATPase isoforms since high amounts of foreign proteins can be produced and some insect cell lines have low levels of endogenous Na,K-ATPase. Recombinant baculoviruses containing the cDNAs for the alpha2, alpha3, and beta1 isoforms of the rat Na,K-ATPase were prepared and used to infect Sf-9 cells, an insect cell line derived from the ovary of the fall armyworm Spodoptera frugiperda. By using this system, Na,K-ATPase alpha2 and alpha3 subunits that were antigenically and electrophoretically indistinguishable from the native subunits were produced. When each subunit is expressed independently in the Sf-9 cells, it is primarily delivered to the plasma membrane. Although the isolated expression of each Na,K-ATPase subunit did not render active Na,K-ATPase molecules, the coexpression of alpha2 or alpha3 with beta1 resulted in catalytically active molecules. This activity could be measured as a ouabain-sensitive ATPase activity or directly demonstrated using either [gamma-P-32]ATP or P-32i to identify the phosphorylated intermediates of the alpha2 and alpha3 isoforms. [H-3]Ouabain binding studies showed that both isoforms are capable of binding the cardiotonic steroid with high affinity, alpha3 being more sensitive to ouabain. These results demonstrate that the baculovirus system is suitable for the expression of the Na,K-ATPase isoforms and should provide a useful method for the characterization of the enzymatic properties of each isoform.