STRONTIUM BINDING TO SARCOPLASMIC-RETICULUM CA-2-ATPASE - SPECTROSCOPIC DIFFERENTIATION OF THE SUBSTEPS INVOLVED()

We investigated the consequences of Sr2+ binding to the transport site s of sarcoplasmic reticulum (SR) Ca2+-ATPase for two fluorescent confo rmational probes located in different regions of the ATPase. Using SR vesicles in which Lys-515 in the ATPase had been previously labeled wi th fluorescein 5'-isothiocyanate (FITC), we found that the Sr2+-induce d a drop in the fluorescein fluorescence of this FITC-labeled ATPase s hifted toward lower Sr2+ concentrations than the Sr2+-induced rise in Trp fluorescence for the same FITC-labeled ATPase. The curve describin g the Sr2+-dependent rise in Trp fluorescence had a characteristic asy mmetric shape, and the changes in Trp fluorescence occurred in paralle l with the activation by Sr2+ of pNPP hydrolysis by the ATPase. Analys is of these results in terms of the simplest scheme describing the seq uential binding of the two Sr2+ ions suggests that under the condition s of these experiments, i.e. at neutral pH in the presence of potassiu m, the Sr2+-induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, wherea s the binding of a single Sr2+ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.