Benzophenone-Sensitized Photooxidation of Sarcoplasmic Reticulum Membranes: Site-Specific Modification of the Ca2+-ATPase

Benzophenone (BP) was used as a photosensitizer to initiate lipid peroxidation in model and native biological membranes at concentrations of BP that do not perturb bilayer structure, as assessed by stearic acid spin label dynamics. Illumination of BP partitioned into sarcoplasmic reticulum membranes (SR) results in an exponential decay of BP and a linear accumulation of conjugated dienes and other products of lipid peroxidation as observed previously for micelles of linoleic acid [Marcovic and Patterson. Photochem. Photobiol. 58:329–334, 1993]. Lipid peroxidation was substantially inhibited in the presence of membrane-spanning proteins in SR compared to protein-free lipid vesicles, suggesting the competitive reaction of the initiator (triplet BP) and BP-derived radical species with protein groups. Modification of the predominant integral membrane protein, the Ca2+-ATPase, was demonstrated by changes in Ca2+-ATPase amino acid composition as well as by its functional inhibition. The rate of calcium transport showed an immediate exponential decay to completion, while calcium-dependent ATPase activity exhibited an initial lag before modest inactivation. These results are consistent with the respective localization of calcium transport sites within membrane-spanning peptides and the ATP-binding site within the cytosolic domain of the Ca2+-ATPase, further suggesting that photosensitization of BP models oxidative stress inside the hydrophobic interior of the SR membrane.