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(Received for publication, April 17, 1995; and in revised form, July 17, 1995) The functional role of serine 775, predicted to be located in
the fifth transmembrane segment of the These data show that the
electronegative oxygen and the small side chain of Ser
Volume 270,
Number 39,
Issue of September 29, pp. 22764-22771, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit of the Na,K-ATPase Selectively Disrupt
K
High Affinity Activation without Affecting
Na
Interaction
subunit of the Na,K-ATPase
(YTLTSNIPE), was studied using site-directed mutagenesis, expression,
and kinetic analysis. Substitutions S775A, S775C, and S775Y were
introduced into an ouabain-resistant 1 sheep isoform and expressed
in HeLa cells. cDNAs carrying substitutions S775C and S775A produced
ouabain-resistant colonies only when extracellular K was increased from 5.4 mM to 10 or 20 mM,
respectively. No ouabain-resistant colonies were obtained for
substitutions S775Y at any tested K
concentration.
Kinetic characterization of S775C and S775A substituted enzymes showed
expression levels higher than control enzyme, reduced V
and turnover, and normal phosphorylation and
high affinity ATP binding. Dephosphorylation experiments indicated that
S775A substituted enzyme is insensitive to ADP but readily
dephosphorylated by K. The K
K values for the activation of the Na,K-ATPase were markedly
altered, with S775C displaying a 13-fold increase and S775A exhibiting
a 31-fold increase. These large changes in the Na,K-ATPase affinity for
K
are consistent with the participation of this amino
acid in binding K
during the translocation of this
cation. Substitutions of Ser
did not change Na
affinity, indicating that this residue is likely not involved in
Na
binding and occlusion.
are required for efficient enzyme function. Moreover, these
results suggest Ser
plays a distinct role in K
transport and not in Na
interactions, revealing
a possible mechanism for the enzymatic differentiation of these cations
by the Na,K-ATPase.
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