Na,k-atpase a subunit containing q905-v930 of gastric h,k-atpase a
preferentially assembles with h,k-atpase b.
Wang, Shyang-Guang, Kurt A. Eakle, Robert Levenson, and Robert A.
Farley.
Department of Physiology and Biophysics, University of Southern
California, Los Angeles, CA 90033, and (Department of Pharmacology,
Milton S. Hershey Medical College, Pennsylvania State University,
Hershey, PA 17033
APStracts 3:0317C, 1996.
Amino acids N886-A911 of the rat Na+/K+-ATPase a3 subunit were
replaced by the corresponding region (Q905-V930) of the rat gastric
H+/K+-ATPase a subunit. The chimera (NGH26) was expressed in yeast
with either the rat Na+/K+-ATPase b1 subunit (Rb1), the rat H+/K+
-ATPase b subunit (HKb), the chimeric b subunit NHb1 (containing the
carboxy-terminal ectodomain of HKb), or the chimeric b subunit HNb1
(containing the carboxy-terminal ectodomain of Rb1). Increased
resistance to trypsin digestion indicated that NGH26 preferentially
assembled with HKb and NHb1 rather than with Rb1 or HNb1. Ouabain
binding also indicated that more functional complexes were assembled
when NGH26 was expressed with HKb or NHb1. These results suggest that
the sequence Q905-V930 interacts with the HKb subunit on the
extracellular side of the cell membrane. The NGH26+HKb complex is
less stable than a3+HKb when heated, and also has a lower binding
affinity for ouabain (Kd = 63 nM) compared to a3+Rb1 or (3+HKb (Kd =
5-10 nM). In contrast, the NGH26+NHb1 complex is thermally as stable
as a3+Rb1 complexes and its ouabain binding affinity (Kd = 11 nM) is
the same as the wild type. These results indicate that the amino
acids Q905-V930 of the rat gastric H+/K+-ATPase a subunit
preferentially associate with the extracellular domain of H+/K+
-ATPase b subunit to form functional pump complexes, and that the
cytoplasmic and/or transmembrane region of the b subunit influences
the stability of the ab complexes.
Received 28 May 1996; accepted in final form 24 September 1996.
APS Manuscript Number C293-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996