Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-d ependent protein kinase (PKA) occurs in intact cells, the purified enzyme i n vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of dif ferent detergents and ligands on the stoichiometry of the phosphorylation a nd activity of Na,K-ATPase from duck salt glands (alpha1 beta1-isoenzyme) w ere comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent rati o in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol P-i/mol of alpha -subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the dete rgent inhibits the Na,K-ATPase. A correlation was found between the inclusi on of P-i into the alpha -subunit and the loss of activity of the Na,K-ATPa se.