AMINO-TERMINAL PROCESSING OF THE CATALYTIC SUBUNIT FROM NA-K+-ATPASE()

The first five amino acids of the catalytic alpha(1)-subunit predicted from its cDNA are not found in purified mammalian Na+-K+-ATPase, sugg esting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site -directed antibody (anti-VGR) specific for the first nine residues of nascent alpha(1) from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobil ity appropriate for the alpha(1)-subunit (100 kDa). Immunoblots of tot al protein from various rat organs, however, revealed no significant b inding, implying that virtually all the alpha(1)-subunit expressed in vivo was modified. We also assessed amino-terminal structure in variou s heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an cll!troponin fusion protein and in insect cells infected with baculovirus containi ng full-length alpha(1) or alpha(1)T. This suggests that modification of the introduced alpha(1) in these expression systems was absent or d ifferent from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha(1) did not reveal significant binding o f the antibody, indicating that the introduced isoform was processed a ppropriately. These results demonstrate that the structure of the alph a(1)-subunit's amino terminus differs among various expression systems . The results further imply that efficient co- or posttranslational pr ocessing of nascent alpha(1) is conserved among various organs within the rat, yet the required modification enzymes are not present in dist ant phyla.