Pre-steady-state phosphorylation of purified Na,K-ATPase from red oute
r medulla of pig kidney was studied at 25-degrees-C and an ample range
of [tau-P-32]ATP concentrations. At 10-mu-M ATP phosphorylation follo
wed simple exponential kinetics reaching after 40 ms a steady level of
0.76 +/- 0.04 nmol of P/mg of protein with k(app) = 73.0 +/- 6.5 s-1.
At 500-mu-M ATP the time course of phosphorylation changed drasticall
y, since the phosphoenzyme reached a level two to four times higher at
a much higher rate (k(app) greater-than-or-equal-to 370 s-1) and in a
bout 40 ms dropped to the same steady level as with 10-mu-M ATP. This
superphosphorylation was not observed in Na,K-ATPase undergoing turnov
er in a medium with Mg2+, Na+, and ATP, suggesting that it required th
e enzyme to be at rest. Superphosphorylation depended on Mg2+ and Naand was fully inhibited by ouabain and FITC. After denaturation the ph
osphoenzyme made by superphosphorylation had the electrophoretic mobil
ity of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was ac
celerated by hydroxylamine. On a molar basis, the stoichiometry of pho
sphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with
1000-mu-M ATP. The results are consistent with the idea that under pro
per conditions every functional Na,K-ATPase unit can accept two, or mo
re, phosphates of rapid turnover from ATP.