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Abstract

Phosphorylation of Na⁺/K⁺‐ATPase by cGMP‐dependent protein kinase (PKG) has been studied in enzymes purified from pig, dog, sheep and rat kidneys, and in Xenopus oocytes. PKG phosphorylates the α‐subunits of all animal species investigated. Phosphorylation of the β‐subunit was not observed. The stoichiometry of phosphorylation estimated for pig, sheep and dog renal Na⁺/K⁺‐ATPase is 3.5, 2.2 and 2.1 mol P_i per mol α‐subunit, respectively. Proteolytic fingerprinting of the pig α1‐subunits phosphorylated by PKG using specific antibodies raised against N‐terminus or C‐terminus reveals that phosphorylation sites are located within the intracellular loop of the α‐subunit between the 35 kDa N‐terminal and 27 kDa C‐terminal fragments. Phosphorylation sites within the α1‐subunit of the purified Na⁺/K⁺‐ATPase do not appear to be easily accessible for PKG since incorporation of P_i requires 0.2% of Triton X‐100. Administration of cGMP and PKG in the presence of 5 mm ATP, which prevents inactivation of the Na⁺/K⁺‐ATPase by detergent, leads to stimulation of hydrolytic activity by 61%. Administration of 50 µm of cGMP or dbcGMP in yolk‐free homogenates of Xenopus oocytes leads to stimulation of ouabain‐dependent ATPase activity by 130–198% and to incorporation of ³³P into the α‐subunit without the detergent. Hence, PKG plays regulatory role in active transmembraneous transport of Na⁺ and K⁺ via phosphorylation of the catalytic subunit of the Na⁺/K⁺‐ATPase.