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, suggesting 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 mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.
Pressley
T A
TA
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Allen
J C
JC
Clarke
C H
CH
Odebunmi
T
T
Higham
S C
SC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D051381:Rats; D016415:Sequence Alignment; D017421:Sequence Analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D000254:Sodium-Potassium-Exchanging ATPase