Reversible changes in the fluorescence energy transfer accompanying formation of reaction intermediates in probe-labeled (Na+,K+)-ATPase.
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.
Taniguchi
K
K
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
Mårdh
S
S
D000085:Acetates / Q000737:chemistry; D000818:Animals; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005453:Fluorescence; D007668:Kidney / Q000201:enzymology; D008301:Maleimides / Q000737:chemistry; D009840:Oligomycins / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013552:Swine
D000085:Acetates; D008301:Maleimides; D009840:Oligomycins; D013439:Sulfhydryl Reagents; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium