J. Biol. Chem., Vol. 255, Issue 4, 1547-1553, Feb, 1980
Characterization of a Mg2+-stabilized state of the (Na+ and K+)-- stimulated adenosine triphosphatase using a fluorescent reporter group
MD Forgac
A Mg2+-induced change of the (Na+ and K+)-stimulated adenosine
triphosphatase (Na+,K+)-ATPase) from Electrophorus electricus was
investigated by kinetics and fluorescence techniques. Binding of Mg2+ to a
low affinity site(s) caused inhibition of (Na+,K+)-ATPase activity, an
effect which was antagonized by both Na+ and ATP. Mg2+ also caused
inhibition of K+-dependent dephosphorylation of the enzyme without
inhibiting either (Na+)-ATPase activity or Na+-dependent phosphorylation.
Mg2+ also induced a 5 to 6% enhancement in the fluorescence intensity of
enzyme labeled with the fluorescent sulfhydryl reagent,
2-(4-maleimidylanilino)naphthalene-6-sulfonate. As in the case of Mg2+
inhibition of activity, the affinity for Mg2+ as an inducing agent for this
effect was significantly reduced by both Na+ and ATP, suggesting that the
same change was being monitored in both cases. The Mg2+ effect was reduced
by both Na+ and ATP, suggesting that the same change was being monitored in
both cases. The Mg2+ effect was reduced in magnitude by ouabain and
prevented by oligomycin, specific inhibitors of the enzyme. In addition, K+
(and cations that substitute for K+ in supporting activity) induced a 3 to
4% enhancement in fluorescence intensity in the presence of Na+, Mg2+, and
ATP, although the K+ and Mg2+ effects appeared to be different on the basis
of their excitation spectra. The K+ effect was inhibited by ouabain and
occurred with a rate greater than the rate of turnover of the enzyme,
permitting its involvement in the catalytic cycle.
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