Location of the Ca(2+)-transport site of Ca(2+)-transporting ATPase of the sarcoplasmic reticulum as determined by analysis of paramagnetic interaction between Gd3+ ions bound at the transport site and membrane-embedded nitroxide spin probes.

Gd3+ ions were bound to the Ca(2+)-transport site of Ca(2+)-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) and their effect on the ESR spectrum of spin-probes, which were attached to specific sites on SR-ATPase and embedded in the membranous lipid at various depths from the surface of the membrane, was studied. Spin-labeled reagents, 1-oxyl-2,2-dimethyl-oxazolidine derivatives of maleimidoethyl-keto stearate, collectively abbreviated as MSL(m,n) were mainly used for labeling SR-ATPase. They have Cm- and Cn-hydrocarbon chains, respectively, on both sides of the spin label, of which the Cm-hydrocarbon chain is located distal to the carboxyl group of the keto stearate moiety. Paramagnetic interaction between Gd3+ and a spin probe was detected by measuring the decrease in the intensity of the ESR signal of the probe. Displacement of Gd3+ from the Ca(2+)-transport site by Ca2+, which had been confirmed previously by using fluorescently labeled SR-ATPase (described in the preceding article), led to a significant reversal of the paramagnetic effect of Gd3+ on MSL(12,3) and MSL(10,5) attached to SR-ATPase. On the other hand, the effect of Gd3+ was not reversed by Ca2+ when SR-ATPase labeled with MSL(1,14) or a spin-label specific for the cytoplasmic domain was used. These results led us to conclude that the Ca(2+)-transport site of SR-ATPase is located in the membranous region of the molecule, but that the site is not very far from the surface of the membrane of the sarcoplasmic reticulum.