J. Biol. Chem., Vol. 263, Issue 17, 7961-7969, Jun, 1988

Rapid 86Rb release from an occluded state of the Na,K-pump reflects the rate of dephosphorylation or dearsenylation

B Forbush 3d
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.

The rapid release of 86Rb from an occluded state of the Na,K-ATPase was studied in a rapid filtration apparatus. In the presence of Mg2+, 86Rb release was found to be stimulated by arsenate and thiophosphate just as it is stimulated by Pi. The affinity of Na,K-ATPase for arsenate is about 4-fold higher than that for Pi and the affinity for thiophosphate is about 5-fold lower than that for Pi. With all three divalent anions, the rates of maximally stimulated 86Rb release were constant between pH 6.5 and 7.5, and decreased between pH 7.5 and 8.5. The affinity for phosphate and thiophosphate increased in the latter pH range, while the affinity for arsenate decreased. The results are not consistent with titration of the divalent anion as the sole determinant of effectiveness in stimulating 86Rb release; thus they suggest that titration of groups on the protein is important. A delay in the rise to the maximum rate of 86Rb release upon stimulation with arsenate is shown to be due to the time required for arsenylation, and from an analysis of the rise and fall of the rate of 86Rb release the rate constants for arsenylation and dearsenylation at pH 7.2 can be estimated; the decay in the rate of 86Rb release when arsenate or phosphate is removed from the solution provides a second method for determination of the dearsenylation rate. The dearsenylation rate constant increases 5-fold from pH 6.1 to 7.5. From the time course of 86Rb release in the presence of Pi we estimate that the rate of dephosphorylation is 50-100 s-1 at pH 6.6 and 20 degrees C; at pH 7-7.5 the rate is too fast to determine. Dimethyl sulfoxide (25%) increases the affinity for arsenate (or phosphate), due to reciprocal changes in arsenylation and dearsenylation rates, and it increases the rate of 86Rb release 2-3 fold. Finally, the level of phosphointermediate formation from 32Pi was determined in the absence and presence of K+: when methanol is used as a denaturant, K+ has only a small effect on the observed level of E-32P, but when trichloroacetic acid is used, K+ is found to reduce the observed level to less than 50% of the control value.
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