Location of the Ca²⁺-Transport Site of Ca²⁺-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

Abstract

Gd³⁺ ions were bound to the Ca²⁺-transport site of Ca²⁺-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-oxy1-2, 2-dimethyl-oxazolidine derivatives of maleimidoethyl-keto stearate, collectively abbreviated as MSL (m, n) were mainly used for labeling SR-ATPase. They have C_m-and C_n-hydrocarbon chains, respectively, on both sides of the spin label, of which the C_m-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 Ca²⁺-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 Ca²⁺-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.