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.
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
K
K
Taniguchi
S
S
Mårdh
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