BIOCHEMICAL-CHARACTERIZATION OF THE SUBUNITS OF THE NA+ K+ ATPASE EXPRESSED IN INSECT CELLS/

The Na+/K+ ATPase is composed of two subunits called alpha and beta ch ains, In insect cells, independently expressed ct and beta chains are localized to intracellular membranes. Sucrose density gradient sedimen tation, crosslinking analysis, and immunoprecipitation of radio-labele d proteins show that the alpha chains expressed alone are in large agg regates of different molecular weights with less than 4% being monomer ic. Analysis by non-reducing SDS-PAGE and immunoblotting show that the beta chains expressed alone are in Triton X-100 insoluble, disulfide- linked aggregates, Go-expression of both subunits in insect cells resu lts 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 determin ed by a biochemical assay. The small amount of beta chain at the plasm a membrane in cells that express both subunits is beyond the limit of detection by the biochemical assay. Immunoprecipitation of Triton X-10 0 soluble alpha chains from radio-labeled cells expressing both subuni ts shows that the alpha chains are mostly in large aggregates containi ng beta chains, These results suggest that, in insect cells, the avail ability of correctly folded beta chains is the rate limiting step in t he assembly of active Na+/K+ ATPase. (C) 1997 Elsevier Science B.V.