AJP - Cell Physiology, Vol 271, Issue 3 C825-C832, Copyright © 1996 by American Physiological Society
Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase
T. A. Pressley, J. C. Allen, C. H. Clarke, T. Odebunmi and S. C. Higham
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
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.
