Amino terminal processing of the catalytic subunit from na+-k+
-atpase.
Pressley, Thomas A., Julius C. Allen, Charlotte H. Clarke, Timothy
Odebunmi, and Sandra C. Higham.
Department of Physiology, Texas Tech University Health Sciences
Center, Lubbock, Texas 79413 and Department of Medicine, Baylor
College of Medicine, Houston, Texas 77030
APStracts 3:0110C, 1996.
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 post-translational 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
polypeptide 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 E. 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 post-translational
processing of nascent [alpha]1 is conserved among various organs
within the rat, yet the required modification enzymes are not present
in distant phyla.
Received 23 October 1995; accepted in final form 15 March 1996.
APS Manuscript Number C643-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 16 April 96