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High-affinity ouabain binding to
Na
Volume 270,
Number 23,
Issue of June 9, pp. 13937-13947, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Structure on the Interaction of
Na
/K
-ATPase Complexes with
Na
A CHIMERIC 
SUBUNIT REDUCES THE Na DEPENDENCE OF PHOSPHOENZYME FORMATION FROM ATP
/K
-ATPase (sodium- and
potassium-transport adenosine triphosphatase (EC 3.6.1.37)) requires
phosphorylation of the
subunit of the enzyme either by ATP or by
inorganic phosphate. For the native enzyme (/1), the
ATP-dependent reaction proceeds about 4-fold more slowly in the absence
of Na than when saturating concentrations of
Na
are present. Hybrid pumps were formed from either
the
1 or the 3 subunit isoforms of
Na/K
-ATPase and a chimeric
subunit containing the transmembrane segment of the
Na/K
-ATPase
1 isoform and the
external domain of the gastric
H/K
-ATPase
subunit
(/NH1 complexes). In the absence of Na,
these complexes show a rate of ATP-dependent ouabain binding from
75-100% of the rate seen in the presence of Na
depending on buffer conditions. Nonhydrolyzable nucleotides or
treatment of ATP with apyrase abolishes ouabain binding, demonstrating
that ouabain binding to
/NH1 complexes requires
phosphorylation of the protein. Buffer ions inhibit ouabain binding by
/NH1 in the absence of Na rather than
promote ouabain binding, indicating that they are not substituting for
sodium ions in the phosphorylation reaction. The pH dependence of
ATP-dependent ouabain binding in the presence or absence of
Na
is similar, suggesting that protons are probably
not substituting for Na
. Hybrid
/NH1 pumps
also show slightly higher apparent affinities (2-3-fold) for ATP,
Na, and ouabain; however, these are not sufficient to
account for the increase in ouabain binding in the absence of
Na
. In contrast to phosphoenzyme formation and ouabain
binding by
/NH1 complexes in the absence of
Na, ATPase activity, measured as release of phosphate
from ATP, requires Na
. These data suggest that the
transition from E
P to E
P
during the catalytic cycle does not occur when the sodium binding sites
are not occupied. Thus, the chimeric subunit reduces or
eliminates the role of Na in phosphoenzyme formation
from ATP, but Na
binding or release by the enzyme is
still required for ATP hydrolysis and release of phosphate.
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