Phosphorylation of the α‐subunits of the Na+/K+‐ATPase from mammalian kidneys and Xenopus oocytes by cGMP‐dependent protein kinase results in stimulation of ATPase activity

Phosphorylation of the α‐subunits of the Na+/K+‐ATPase from mammalian kidneys and Xenopus oocytes by cGMP‐dependent protein kinase results in stimulation of ATPase activity

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 stoi...

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Bibliographic Details
Published in:European journal of biochemistry Vol. 260; no. 3; pp. 904 - 910
Main Authors: Fotis, Heike, Tatjanenko, Liliya V., Vasilets, Larisa A.
Format: Journal Article
Language:English
Published: Oxford, UK Blackwell Science Ltd 01-03-1999
Subjects:
Adenosine Triphosphatases - metabolism
Animals
cGMP
Cyclic GMP-Dependent Protein Kinases - metabolism
Dogs
Enzyme Activation
K+‐ATPase
kidney
Kidney - enzymology
Na+
Oocytes - enzymology
Phosphorylation
PKG
Rats
Sheep
Sodium-Potassium-Exchanging ATPase - metabolism
Swine
Xenopus
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Summary: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 Pi 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 Pi 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.
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ISSN:0014-2956
1432-1033
DOI:10.1046/j.1432-1327.1999.00237.x