The metal sites on sarcoplasmic reticulum membranes that bind lanthanide ions with the highest affinity are not the ATPase Ca2+ transport sites.
We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.
Henao
F
F
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Badajoz, Spain.
Orlowski
S
S
Merah
Z
Z
Champeil
P
P
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D008674:Metals, Rare Earth / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013379:Substrate Specificity
D002412:Cations; D008670:Metals; D008674:Metals, Rare Earth; D000252:Calcium-Transporting ATPases