J. Biol. Chem., Vol. 255, Issue 12, 5724-5727, Jun, 1980
Inhibition by vanadate of the reactions catalyzed by the (Na+ + K+)- stimulated ATPase. A transient state kinetic characterization
AS Hobbs, JP Froehlich and RW Albers
The interaction of vanadate with the (Na+ + K+)-stimulated ATPase from
electric organ was investigated using the acid quench-flow technique. At 21
degrees C, incubation of the enzyme with 1.3 to 1.6 muM vanadate in the
presence of 75 mM Na+ and 25 mM K+ strongly inhibits phosphorylation by
ATP. Enzyme activity remaining under these conditions shows no change in
the apparent rates of phosphorylation or dephosphorylation, although
effects were noted which suggest that vanadate increases the reverse rate
of dephosphorylation. Ten micromolar vanadate, sufficient to inhibit the
(Na+ + K+)-stimulated ATPase by more than 98%, has no effect on
phosphorylation in the presence of Na+ alone. Phosphoenzyme formed in the
presence of Na+ and K+ consists of rapidly and slowly decaying components
which differ in sensitivity to vanadate. Up to 2 muM vanadate suppresses
predominantly the rapidly decaying phosphoenzyme, while at higher
concentrations vanadate inhibits both the rate and level of formation of
the slowly decaying phosphoenzyme. These results indicate that vanadate is
a useful reagent for distinguishing between these two phosphorylation
reactions.
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