Phosphorylation/dephosphorylation of reconstituted shark Na+,K+-ATPase: one phosphorylation site per αβ protomer

AbstractIn the present investigation reconstitution of Na+,K+-ATPase increases the number of phosphorylation sites (EP) of solubilized enzyme from 4.2 ± 0.3 nmol/mg to 6.9 ± 0.6 nmol/mg. The latter figure corresponds to one phosphorylation site per αβ-protomer. A cholesterol content > 10 mol% in the liposome bilayer and a high extracellular [Na+ are necessary to obtain this high value. Spontaneous dephosphorylation after maximum phosphorylation in Na+ is biphasic both in solubilized enzyme and after reconstitution. The rate of dephosphorylation compares with the specific hydrolytic Na+-ATPase activity measured at exactly identical conditions for all three preparations assuming parallel dephosphorylation of at least two phosphointermediates. The distribution of EP-species is found to vary among the three enzyme preparation used, i.e., membrane bound, solubilized, and reconstituted Na+,K+-ATPase, however in all the equilibrium is strongly poised away from the E1P-form