A Spin-Label and Hydrogen-Deuterium Exchange Reaction Kinetics Study of Protein-Lipid Interactions in Lipid-Replaced Ca2+-ATPase of Rabbit Skeletal Muscle Sarcoplasmic Reticulum1

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Abstract
The Ca2+-ATPase of sarcoplasmic reticulum from rabbit skeletal muscle was incorporated into vesicles made from dimyristoylphosphatidylcholine or dipalmitoylphosphatidyicholine. The Ca2+-ATPase activity of these reconstituted membranes became appreciable above 20°C and 30°C, respectively, in accord with the results of previous investigators. Measurement by the spin-labeling technique of the fluidity of the bulk lipid revealed the gel-to-liquid crystalline phase transition at 29°C and 39°C, respectively, while the fluidity of the boundary lipid in both samples was found to be low throughout the temperature range studied. The rotational mobility of the Ca2+-ATPase protein in both samples, measured by saturation transfer electron spin resonance, was also very low throughout the temperature range studied and its temperature-dependence did not show any break or jump corresponding to the phase transition of the bulk lipid. On the other hand, the structural fluctuation of the Ca2+-ATPase protein in dimyristoylphosphatidylcholinerecombinant, measured in terms of hydrogen-deuterium exchange reaction kinetics, showed a jump at about 27°C, apparently in accordance with the phase transition of the bulk lipid. Results obtained in this study suggested that the Ca2+-ATPase protein molecules are in an aggregated state in these reconstituted membranes and that the Ca2+-ATPase activity is neither directly correlated to the fluidity of the boundary lipid nor to the rotational mobility of the Ca2+-ATPase, contrary to the suggestions of previous investigators (Hesketh et al. (1976) Biochemistry 15, 4145–4151; Hidalgo et at. (1978) J. Biol. Chem. 253, 6879–6887).

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