title: Strontium binding to sarcoplasmic reticulum Ca(2+)-ATPase. Spectroscopic differentiation of the substeps involved.
abstract: We investigated the consequences of Sr2+ binding to the transport sites of sarcoplasmic reticulum (SR) Ca(2+)-ATPase for two fluorescent conformational probes located in different regions of the ATPase. Using SR vesicles in which Lys-515 in the ATPase had been previously labeled with fluorescein 5'-isothiocyanate (FITC), we found that the Sr(2+)-induced a drop in the fluorescein fluorescence of this FITC-labeled ATPase shifted toward lower Sr2+ concentrations than the Sr(2+)-induced rise in Trp fluorescence for the same FITC-labeled ATPase. The curve describing the Sr(2+)-dependent rise in Trp fluorescence had a characteristic asymmetric shape, and the changes in Trp fluorescence occurred in parallel with the activation by Sr2+ of pNPP hydrolysis by the ATPase. Analysis of these results in terms of the simplest scheme describing the sequential binding of the two Sr2+ ions suggests that under the conditions of these experiments, i.e. at neutral pH in the presence of potassium, the Sr(2+)-induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, whereas the binding of a single Sr2+ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.
authors.affiliation: Unité de Recherche Associée 1290 (Centre National de la Recherche Scientifique), Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
authors.forename: S
authors.initials: S
authors.lastname: Orlowski
authors.forename: P
authors.initials: P
authors.lastname: Champeil
mesh_terms: D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013324:Strontium / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D014364:Tryptophan; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D013324:Strontium