Characterization of ruthenium red-binding sites of the Ca2+-ATPase from sarcoplasmic reticulum and their interaction with Ca2+-binding sites

Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5′-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites.