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  • Title: Reversible changes in the fluorescence energy transfer accompanying formation of reaction intermediates in probe-labeled (Na+,K+)-ATPase.
    Author: Taniguchi K, Mårdh S.
    Journal: J Biol Chem; 1993 Jul 25; 268(21):15588-94. PubMed ID: 8393448.
    Abstract:
    A preparation of pig kidney Na+,K(+)-ATPase showed changes in fluorescence energy transfer between probes bound to the alpha-subunit. Excitation (305 nm) of an N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) probe, which was covalently bound to Cys-964, and excitation (470 nm) of a fluorescein 5'-isothiocyanate (FITC) probe at Lys-501 gave different FITC fluorescence intensity changes at 520 nm in BIPM-FITC doubly labeled enzyme accompanying formation of reaction intermediates. Addition of acetyl phosphate to a Na(+)-bound enzyme (NaE1) to accumulate acetate-sensitive phosphoenzyme (E1P) induced a faster and greater FITC fluorescence decrease when excited at 470 nm than at 305 nm. An oligomycin-sensitive transition of E1P to K(+)-sensitive phosphoenzyme (E2P) was also accompanied by a larger FITC fluorescence decrease when excited at 470 nm. The addition of K+ to [32P]E2P to form a K(+)-bound enzyme (KE2) induced a rapid dephosphorylation (140/s) with both a slow rate of FITC fluorescence increase and a larger fluorescence increase when excited at 470 nm. The addition of Na+ to KE2 induced both a slow increase of FITC fluorescence and a larger fluorescence increase when excited at 470 nm. The data suggest that fluorescence energy transfer from the BIPM to the FITC probe accompanies the processes of migration of Na+ and K+ in the pump molecules. The dynamic fluorescence changes after phosphorylation and dephosphorylation seem to reflect changes in the binding state or the process of migration of these ions.
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