Phosphorylation/dephosphorylation of reconstituted shark Na+,K(+)-ATPase: one phosphorylation site per alpha beta...

In 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 alpha beta-promoter. 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.