Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na+ + K+)-activated ATPase.
The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50-65 degrees C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. Thermodynamic parameters (delta H degree and delta S degree) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and AMP least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. The nucleotide specificity of the low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P greater than ATP greater than ADP greater than AMP. The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. It is inferred that the low-affinity site is located on the enzyme, more specifically its alpha-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D010042:Ouabain / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013816:Thermodynamics
D000266:Adenylyl Imidodiphosphate; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D005033:Ethylmaleimide