LOW AFFINITY SUPERPHOSPHORYLATION OF THE NA,K-ATPASE BY ATP

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.