Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk-cells with DNA encoding the beta-subunit of an avian brain sodium pump.
A cDNA encoding the beta-subunit of the (Na+ + K+)-ATPase was cloned from a chicken brain cDNA library, and its nucleotide sequence was determined. High cross-species sequence homologies were found both in coding and noncoding regions. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk-cells. The avian beta-subunit was expressed on the cell surface (1-8 X 10(5) molecules/cell) complexed with alpha-subunits of the murine (Na+ + K+)-ATPase. In the hybrid system there was rapid assembly of subunits, post-translational N-glycosylations of the beta-subunit at its three Asn-X-Ser (or Thr) positions, and modification of high mannose oligosaccharides to complex type. Avian beta-subunits expressed in the mouse cells had an apparent molecular weight of about 55,000 as compared with 47,000 in avian cells, due to post-translational modifications, presumably differences in complex oligosaccharides. Despite the high number of interspecies hybrid (Na+ + K+)-ATPase molecules, the cells had none of the high affinity ouabain binding sites (KD = 2 X 10(-7) M) characteristic of avian cells, consistent with the view that the ouabain binding site is located largely or exclusively on the alpha-subunit and is not greatly affected by alpha-beta interaction.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002642:Chick Embryo; D002645:Chickens; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D006031:Glycosylation; D051379:Mice; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D010957:Plasmids; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D004274:DNA, Recombinant; D009844:Oligosaccharides; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase