Immobilization of a spin-labeled fatty acid chain covalently attached to calcium(2+)-ATPase from sarcoplasmic reticulum suggests an oligomeric structure

A spin-labeled fatty acid chain (16-doxylstearic acid), linked by an ester bond to a maleimide residue, was covalently attached to the Ca2+-ATPase of rabbit sarcoplasmic reticulum. Three different hydrophobic environments of Ca2+-ATPase were investigated by electron spin resonance: (i) native sarcoplasmic reticulum, (ii) reconstituted vesicles obtained by exchange of the endogenous lipids with egg lecithin, and (in) Ca2+-ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether (C12E8). All spectra were composite, with a major component corresponding to strongly immobilized probe (called “immobilized component”). The percentage of immobilized component seemed to decrease with increasing temperature or in the presence of a small amount of C12E8, under nonsolubilizing conditions. Quantitation of the percentage of immobilized component was performed by linear combination of pairs of appropriately selected spectra, for which it could be assumed that changes predominantly were caused by differences in the proportion of immobile and mobile components and not by changes in line shape. This was the case either by a small variation in temperature or by addition of small amounts of C12E8. The immobilized component generated by the combination of such spectra is characterized by a large splitting with only a moderate temperature dependence (from 66 G around 4 °C to 63 G around 35 °C). Partial immobilization persisted after solubilization of ATPase with C12E8. Ultracentrifugation studies demonstrated that the immobilized component was associated with the presence of oligomeric ATPase in the solution. Saturation transfer electron spin resonance revealed a rotational correlation time of 5 X 10-5 s (at 15 °C) for the immobilized component in complexes with egg phosphatidylcholine. This value is close to the rotational correlation time of the protein as determined by saturation transfer using a short-chain maleimide spin-label. We propose that the immobilized component arises from lipid chains trapped in protein oligomers, which exist in native sarcoplasmic reticulum. Partial dissociation of the oligomers may be induced by a rise of temperature or by perturbation of membrane structure with a low concentration of detergent.