In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 97, No. 21 ( 2000-10-10), p. 11209-11214
Abstract:
Na + ,K + -ATPase and gastric
H + ,K + -ATPase are two related enzymes that are
responsible for active cation transport. Na + ,K + -ATPase activity is inhibited
specifically by ouabain, whereas H + ,K + -ATPase
is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na + ,K + -ATPase are
responsible for ouabain binding, we prepared chimeras in which small parts of the α-subunit of H + ,K + -ATPase
were replaced by their counterparts of the α 1 -subunit of
rat Na + ,K + -ATPase. A chimeric enzyme in which
transmembrane segments 5 and 6 of H + ,K + -ATPase
were replaced by those of Na + ,K + -ATPase could
form a phosphorylated intermediate, but hardly showed a K + -stimulated dephosphorylation reaction. When
transmembrane segments 3 and 4 of Na + ,K + -ATPase
were also included in this chimeric ATPase, K + -stimulated
dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this
chimeric enzyme, HN34/56, had obtained a high-affinity ouabain-binding site, whereas the rat
Na + ,K + -ATPase, from which the hairpins
originate, has a low affinity for ouabain. The low affinity of the rat Na + ,K + -ATPase previously had been attributed to
the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34/56 chimera, the M1/M2 loop, however,
originates from H + ,K + -ATPase, which has two
polar uncharged amino acids on this position. Placement of two charged amino acids in the M1/M2 loop of chimera HN34/56 results in a
decreased ouabain affinity. This indicates that although the M1/M2 loop affects the ouabain affinity, binding occurs when the M3/M4 and
M5/M6 hairpins of Na + ,K + -ATPase are present.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.200109597
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2000
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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