Influence of Na+ on conformational states in membrane-bound renal Na,K-ATPase.
Conformational states of the Na,K-ATPase and rates of transition between these are studied using the fluorescent dye eosin as a marker for the Na+ form (E1) and occlusion of 86Rb+ as a marker for the K+ form (E2). The aim of the present paper is to propose that the E1 form of the Na,K-ATPase can be liganded with a number of Rb+ and Na+ ions and that only some of these E1 forms bind eosin and thus probably nucleotides with high affinity. Experiments are performed with Na,K-ATPase isolated from pig kidney. Binding of eosin occurs only when Na+ is present at millimolar concentrations, and the observed rate of binding is slow when Rb+ is present. The rate of eosin binding after a sudden increase in the Na+ concentration is about the same as the rate of deocclusion of Rb+, suggesting that eosin monitors the rate of the E2 to E1 transition. Titrations of eosin fluorescence with Na+ indicate that binding of more than one Na+ occurs when high-affinity eosin binding takes place. With 0.05 mM RbCl and 4 mM NaCl present, the Na,K-ATPase is a mixture of at least two enzyme species which do not bind eosin with high affinity. One species is the E2 form with Rb+ occluded, and transition of this form to E1 gives rise to a small observed rate constant for eosin binding when the Na+ concentration is suddenly increased to about 25 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Esmann
M
M
Department of Biophysics, University of Aarhus, Denmark.
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology*; D004801:Eosine Yellowish-(YS); D005456:Fluorescent Dyes; D007679:Kidney Medulla / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D012414:Rubidium Radioisotopes / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013552:Swine
D005456:Fluorescent Dyes; D012414:Rubidium Radioisotopes; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D004801:Eosine Yellowish-(YS)