A remarkably stable phosphorylated form of Ca2+-ATPase prepared from Ca2+-loaded and fluorescein isothiocyanate-labeled sarcoplasmic reticulum vesicles

After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase h as been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phos phorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981 ) FEES Lett. 123, 131-136), We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this lo w fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore, This species is a Ca2+-free phosp horylated species. The kinetics of Ca2+ binding to it indicates that its tr ansport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsic ally transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP, The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase speci es remains stable after addition of thapsigargsn in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remark able stability of this phosphoenzyme species and the changes in FITC spectr oscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2 +-ATPase, and the possible formation of a transition statelike conformation with a compact cytosolic head. These findings might open a path toward str uctural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.