PHOSPHORYLATION DEPHOSPHORYLATION OF RECONSTITUTED SHARK NA-ATPASE - ONE PHOSPHORYLATION SITE PER ALPHA-BETA PROTOMER(,K+)

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 correspon ds to one phosphorylation site per alpha beta-protomer. A cholesterol content > 10 mol% in the liposome bilayer and a high extracellular [Na +] are necessary to obtain this high value. Spontaneous dephosphorylat ion after maximum phosphorylation in Na+ is biphasic both in solubiliz ed enzyme and after reconstitution. The rate of dephosphorylation comp ares with the specific hydrolytic Na+-ATPase activity measured at exac tly identical conditions for all three preparations assuming parallel dephosphorylation of at least two phosphointermediates. The distributi on of EP-species is found to vary among the three enzyme preparation u sed, i.e., membrane bound, solubilized, and reconstituted Na+,K+-ATPas e, however in all the equilibrium is strongly poised away from the E(1 )P-form.