Blanco G, Xie Z J, Mercer R W
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1824-8. doi: 10.1073/pnas.90.5.1824.
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 alpha 2, alpha 3, and beta 1 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 alpha 2 and alpha 3 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 alpha 2 or alpha 3 with beta 1 resulted in catalytically active molecules. This activity could be measured as a ouabain-sensitive ATPase activity or directly demonstrated using either [gamma-32P]ATP or 32Pi to identify the phosphorylated intermediates of the alpha 2 and alpha 3 isoforms. [3H]Ouabain binding studies showed that both isoforms are capable of binding the cardiotonic steroid with high affinity, alpha 3 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.
已鉴定出钠钾-ATP酶α和β亚基的多种同工型。由于大多数组织表达多种同工型，且尚未实现针对每种同工型的纯化技术，因此阐明它们的作用变得复杂。杆状病毒表达系统利用苜蓿银纹夜蛾杆状病毒感染昆虫细胞，是研究钠钾-ATP酶同工型的理想系统，因为可以产生大量外源蛋白，并且一些昆虫细胞系内源性钠钾-ATP酶水平较低。制备了含有大鼠钠钾-ATP酶α2、α3和β1同工型cDNA的重组杆状病毒，并用于感染Sf-9细胞，这是一种源自草地贪夜蛾卵巢的昆虫细胞系。通过使用该系统，产生了在抗原性和电泳上与天然亚基无法区分的钠钾-ATP酶α2和α3亚基。当每个亚基在Sf-9细胞中独立表达时，它主要被转运到质膜。虽然每个钠钾-ATP酶亚基的单独表达并未产生有活性的钠钾-ATP酶分子，但α2或α3与β1的共表达产生了具有催化活性的分子。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-32P]ATP或32Pi直接证明，以鉴定α2和α3同工型的磷酸化中间体。[3H]哇巴因结合研究表明，两种同工型都能够以高亲和力结合强心甾，α3对哇巴因更敏感。这些结果表明，杆状病毒系统适用于钠钾-ATP酶同工型的表达，应该为表征每种同工型的酶学性质提供一种有用的方法。
