Liu J Y, Guidotti G
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Biochim Biophys Acta. 1997 Oct 20;1336(3):370-86. doi: 10.1016/s0304-4165(96)00153-5.
The Na+/K+ ATPase is composed of two subunits called alpha and beta chains. In insect cells, independently expressed alpha and beta chains are localized to intracellular membranes. Sucrose density gradient sedimentation, crosslinking analysis, and immunoprecipitation of radio-labeled proteins show that the alpha chains expressed alone are in large aggregates of different molecular weights with less than 4% being monomeric. Analysis by non-reducing SDS-PAGE and immunoblotting show that the beta chains expressed alone are in Triton X-100 insoluble, disulfide-linked aggregates. Co-expression of both subunits in insect cells results in only a small fraction (less than 15%) of the alpha chains being assembled as the active recombinant enzyme, with at least 22% of the active recombinant enzyme localized to the plasma membrane as determined by a biochemical assay. The small amount of beta chain at the plasma membrane in cells that express both subunits is beyond the limit of detection by the biochemical assay. Immunoprecipitation of Triton X-100 soluble alpha chains from radio-labeled cells expressing both subunits shows that the alpha chains are mostly in large aggregates containing beta chains. These results suggest that, in insect cells, the availability of correctly folded beta chains is the rate limiting step in the assembly of active Na+/K+ ATPase.
钠钾ATP酶由两个亚基组成，分别称为α链和β链。在昆虫细胞中，单独表达的α链和β链定位于细胞内膜。蔗糖密度梯度沉降、交联分析以及对放射性标记蛋白的免疫沉淀表明，单独表达的α链存在于不同分子量的大聚集体中，单体形式的不到4%。非还原SDS-PAGE和免疫印迹分析表明，单独表达的β链存在于不溶于Triton X-100的二硫键连接的聚集体中。在昆虫细胞中共表达两个亚基时，只有一小部分（不到15%）的α链组装成有活性的重组酶，通过生化分析测定，至少22%的有活性重组酶定位于质膜。在同时表达两个亚基的细胞中，质膜上少量的β链超出了生化分析的检测限。对表达两个亚基的放射性标记细胞中Triton X-100可溶性α链进行免疫沉淀表明，α链大多存在于含有β链的大聚集体中。这些结果表明，在昆虫细胞中，正确折叠的β链的可用性是活性钠钾ATP酶组装的限速步骤。
