Role of Mg2+ ions in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na+,K(+)-ATPase.
The role of Mg2+ in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na,K-ATPase has been studied by stopped-flow fluorometry. K+ causes a fluorescence quench that is reversed by Na+. The principal experimental observations are as follows: (1) Mg2+ decreases the apparent affinity of the enzyme for K+ but does not affect the maximum rate of the K+ quench. (2) The amplitude of the K+ quench depends hyperbolically on the K+ concentration, and the maximum amplitude is unaffected by the Mg2+ concentration. (3) The rate at which Na+ reverses the K+ quench depends inversely on the Mg2+ concentration. (4) The amplitude of the Na+ reversal also decreases with increasing Mg2+ concentration. The data are quantitatively explained by a model that assumes only two enzyme conformations, detectable by their fluorescence emission. Mg2+ increases Kd for K+ from 14 to 223 mM. At 22 degrees C, Kd = 0.16 mM for Mg2+ dissociation from E1, and the heat of Mg2+ binding, delta H degrees, is 11.4 kcal mol-1. Kd is more than an order of magnitude larger for Mg2+ dissociation from E2K. Mg2+ binding does not affect the forward (E1K-->E2K) rate constant (kf), but decreases the reverse rate constant (kr) thus increasing the equilibrium constant for the reaction (Kc = kf/kr) 6-fold. Therefore, Mg2+ is not directly involved in the conformational transition, but the study supports proposals that Mg2+ binding and release may help to regulate the transport cycle by shifting the distribution of enzyme between E1 and E2 conformers.
Smirnova
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IN
Department of Medicine, University of California, Los Angeles School of Medicine.
Faller
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LD
D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000502:physiology*; D008956:Models, Chemical; D011188:Potassium; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D011188:Potassium