(Na + + K +)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na +-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant

Abstract

(1) The dephosphorylation kinetics of acid-stable phosphointermediates of (Na + + K +)-ATPase from ox brain, ox kidney and pig kidney was studied at 0°C. (2) Experiments performed on brain enzyme phosphorylated at 0°C in the presence of 20–600 mM Na +, 1 mM Mg 2+ and 25 μM [γ- 32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na + concentration, all phosphoenzyme is either ADP- or K +-sensitive. (3) After phosphorylation of kidney enzymes at 0°C with 1 mM Mg 2+, 25 μM [γ- 32P]ATP and 150–1000 mM Na + the amounts of ADP- and K +-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.5 mM ADP or 1 mM ATP + 20 mM K +. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na +, after addition of 1 mM ATP + 20 mM K + is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E 1 ≈ P(Na 3) as intermediate of (Na + + K +)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K +-sensitive phosphoenzymes is greater than E tot. (4) In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k −1, is obtained. k −1 varies between 1 and 4 s −1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na + or Tris +. (5) The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na +-ATPase activity.