DeTomaso A W, Xie Z J, Liu G, Mercer R W
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1993 Jan 15;268(2):1470-8.
Deciphering the roles of the individual subunits of the heteromeric Na,K-ATPase in the structure, function, and assembly of this enzyme has been complicated because most expression systems have endogenous levels of Na,K-ATPase activity. This problem has become even more significant in light of the recent identification of multiple isoforms for both the alpha and beta subunits. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, affords two distinct advantages for the expression of the Na,K-ATPase; some insect cells have little or no levels of Na,K-ATPase, and baculovirus-infected cells produce extremely high levels of foreign protein. We have made two separate recombinant baculoviruses containing the rodent alpha 1 or beta 1 cDNAs and used them to infect the insect cell line Sf-9. The infected Sf-9 cells produce Na,K-ATPase subunit protein on the order of 5-10 micrograms of protein/ml of cultured cells. The rodent alpha 1 polypeptide produced in the Sf-9 cells is indistinguishable electrophoretically and antigenically from the native subunit. The expressed beta 1 subunit is also antigenically identical but has a higher electrophoretic mobility due to differential glycosylation by the infected Sf-9 cell. In contrast to other systems, when expressed alone, each individual Na,K-ATPase subunit is targeted to the infected Sf-9 plasma membrane. In contrast, when infected with a virus that induces the heavy chain of murine IgG, the infected Sf-9 cell retains the polypeptide in the endoplasmic reticulum. However, when both IgG light and heavy chains are expressed, the polypeptides are properly processed and secreted. When the Na,K-ATPase alpha 1 and beta 1 polypeptides are simultaneously expressed, they form detergent-resistant complexes that are functional. Ouabain-sensitive ATPase activity on the order of 5 mumol Pi/mg/h in infected Sf-9 membranes was dependent on the expression of both the alpha 1 and beta 1 subunits. Sodium-dependent phosphorylated intermediates were detected that were potassium- and ouabain-sensitive. No increase in ouabain-sensitive activity or phosphorylated intermediates was detected when either subunit was expressed alone. The alpha 1 beta 1-coinfected cells were also able to transport ions, as detected in 86Rb uptake experiments. Thus, the recombinant Na,K-ATPase expressed in insect cells is biologically active and is suitable for structural and functional analysis.
由于大多数表达系统都有内源性的钠钾ATP酶活性，因此要阐明异源钠钾ATP酶中各个亚基在该酶的结构、功能和组装中的作用变得很复杂。鉴于最近发现α和β亚基都有多种亚型，这个问题变得更加突出。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，在表达钠钾ATP酶方面具有两个明显的优势；一些昆虫细胞几乎没有或完全没有钠钾ATP酶活性，并且杆状病毒感染的细胞能产生极高水平的外源蛋白。我们制备了两种分别含有啮齿动物α1或β1 cDNA的重组杆状病毒，并用于感染昆虫细胞系Sf-9。被感染的Sf-9细胞产生钠钾ATP酶亚基蛋白的量约为每毫升培养细胞5-10微克蛋白。在Sf-9细胞中产生的啮齿动物α1多肽在电泳和抗原性上与天然亚基无法区分。表达的β1亚基在抗原性上也相同，但由于被感染的Sf-9细胞的糖基化差异，其电泳迁移率更高。与其他系统不同，单独表达时，每个钠钾ATP酶亚基都定位于被感染的Sf-9细胞膜。相反，当用诱导鼠IgG重链的病毒感染时，被感染的Sf-9细胞将多肽保留在内质网中。然而，当同时表达IgG轻链和重链时，多肽会被正确加工并分泌。当钠钾ATP酶α1和β1多肽同时表达时，它们会形成具有功能的抗去污剂复合物。在被感染的Sf-9细胞膜中，哇巴因敏感的ATP酶活性约为5微摩尔无机磷/毫克/小时，这依赖于α1和β1亚基的表达。检测到了钠依赖性磷酸化中间体，它们对钾和哇巴因敏感。单独表达任何一个亚基时，未检测到哇巴因敏感活性或磷酸化中间体的增加。如在86Rb摄取实验中所检测到的，α1β1共感染的细胞也能够运输离子。因此，在昆虫细胞中表达的重组钠钾ATP酶具有生物学活性，适用于结构和功能分析。
