Steady-state levels of phosphorylated intermediates of (Na,K)-ATPase monitored with oligomycin and anthroylouabain.
We present experiments designed to test whether or not the ADP-sensitive (E1P) and the K-sensitive (E2P) phosphoenzyme forms of (Na,K)-ATPase are intermediates of ATP hydrolysis in the presence of Na and K. We have used the apparent rate constant of anthroylouabain binding, k' on, and its inhibition by oligomycin to monitor the steady-state levels of E2P and E1P, respectively. We have measured k' on in purified dog kidney (Na,K)-ATPase at 37 degrees C as a function of Na, K, and MgATP or Mg plus inorganic phosphate in the presence and absence of oligomycin. In the presence of 35 mM Na and no K, k' on was saturated with [ATP] greater than 25 microM, and oligomycin (10 microM) inhibited less than 10%. Inhibition by oligomycin increased when [Na] was increased. At 1.9 M Na oligomycin inhibited k' on and equilibrium binding of anthroylouabain with Ki = 136 nM. Oligomycin had no effect on k' on in the presence of Mg + inorganic phosphate. In the presence of 35 mM Na and 4.5 mM K, k' on was much slower, inhibition by oligomycin increased, and ATP stimulated both k' on and inhibition by oligomycin with K0.5 greater than 0.1 mM. From the k' on measurements in the presence and absence of K, we estimated that the steady-state level of E2P was between 4 and 15% of the enzyme with 25 microM to 6.5 mM ATP, 35 mM Na, and 4.5 mM K at 37 degrees C. For these conditions we estimated the rate constant of E2P hydrolysis to be 46,000 (+/- 1,000) min-1. These results are contrary to the predictions derived from a model in which acid-stable phosphoenzymes are not intermediates of ATP hydrolysis in the presence of Na and K. They are fully consistent with a model in which E1P and E2P are intermediates of ATP hydrolysis coupled to transport of Na and K.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000873:Anthracenes / Q000378:metabolism*; D004285:Dogs; D005456:Fluorescent Dyes / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D009840:Oligomycins / Q000494:pharmacology*; D010042:Ouabain / Q000031:analogs & derivatives* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D000873:Anthracenes; D005456:Fluorescent Dyes; D009840:Oligomycins; D010042:Ouabain; C013814:anthroylouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium