Changes in Fluorescence Energy Transfer between Sulfhydryl Fluorescent Residues during Ouabain Sensitive Na⁺, K⁺-ATP Hydrolysist

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Na⁺, K⁺-ATPase from pig kidney was sequentially modified with two different sulfhydryl fluorescent reagents, N-[p-(2-benzimidazolyl)phenyl] maleimide (BIPM) and N-[7-dimethylamino 4-coumarinyl]maleimide (DACM). The preparation thus obtained contained 3 and 2 moles of each residue in the α-chain. When the BIPM residues were excited at 313 nm, ouabain sensitive decrease and increase in the fluorescence intensity at not only 365 nm (BIPM fluorescence) but also 455 nm (DACM fluorescence) were observed, which were dependent on the amounts of reaction intermediates accumulated. When DACM residues were excited directly at 390 nm, only the decrease in the fluorescence intensity was observed irrespective of the intermediates accumulated. The data suggest that at least two DACM residues which differently change their microenvironments during ouabain sensitive Na⁺, K⁺-ATPase reaction are present. One is located close enough and the other is located too far to accept the energy from BIPM residue(s) in the three dimensional structure of Na⁺, K⁺-ATPase. Addition of sodium dodecyl sulfate (SDS) remarkably inhibited the energy transfer from BIPM to DACM residues. Limited proteolysis suggested that BIPM residues are located mainly in the peptides which are assumed to contain ATP binding sites and that DACM residues are located near the phosphorylation sites.