Structural dynamics and oligomeric interactions of Na+,K(+)-ATPase as monitored using fluorescence energy transfer.
The oligomeric nature of the purified lamb kidney Na+,K(+)-ATPase was investigated by measuring the fluorescence energy transfer between catalytic (alpha) subunits following sequential labeling with fluorescein 5'-isothiocyanate (FITC) and erythrosin 5'-isothiocyanate (ErITC). Although these two probes had different spectral responses upon reaction with the enzyme, our studies suggest that a sizeable proportion of their binding occurs at the same ATP protectable, active site domain of alpha. Fluorescence energy transfer (FET) from donor (FITC) to acceptor (ErITC) revealed an apparent 56 A distance between the putative ATP binding sites of alpha subunits, which is consistent with (alpha beta)2 dimers rather than randomly spaced alpha beta heteromonomers. In this work, methods were introduced to eliminate the contribution of nonspecific probe labeling to FET values and to determine the most probable orientation factor (K2) for these rigidly bound fluorophores. FET measurements between anthroylouabain/ErITC, 5'-iodoacetamide fluorescein (5'IAF)/ErITC, and TNP-ATP/FITC, donor/acceptor pairs were also made. Interestingly, none of these distances were affected by ligand-dependent changes in enzyme conformation. These results and those from electron microscopy imaging (Ting-Beall et al. 1990. FEBS Lett. 265:121) suggest a model in which ATP binding sites of (alpha beta)2 dimers are 56 A apart, and reside 30 A from the intracellular surface of the membrane contiguous with the phosphorylation domain.
Amler
E
E
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Abbott
A
A
Ball
W J
WJ
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D004735:Energy Transfer; D004923:Erythrosine / Q000031:analogs & derivatives; D016650:Fluorescein-5-isothiocyanate; D017879:Isothiocyanates; D007668:Kidney / Q000201:enzymology; D015394:Molecular Structure; D011487:Protein Conformation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D012995:Solubility; D013050:Spectrometry, Fluorescence
D017879:Isothiocyanates; C022232:erythrosine isothiocyanate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D004923:Erythrosine