Ion selectivity of the cytoplasmic binding sites of the Na,K-ATPase: I. Sodium binding is associated with a conformational rearrangement.
To investigate Na+ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na+-titration experiments were performed using two fluorescent dyes, RH421 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na+ in the presence of various Mg2+ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na+ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na+ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na+ ions and followed by the conformational transition E1/E2 of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na+ ions are bound.
Schneeberger
A
A
Department of Biology, University of Konstanz, Fach M635, D-78457 Konstanz, Germany.
Apell
H J
HJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017136:Ion Transport; D008274:Magnesium / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011726:Pyridinium Compounds / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013343:Styrenes / Q000378:metabolism
D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D011188:Potassium