A spin-label and hydrogen-deuterium exchange reaction kinetics study of protein-lipid interactions in lipid-replaced Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.
The Ca2+-ATPase of sarcoplasmic reticulum from rabbit skeletal muscle was incorporated into vesicles made from dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The Ca2+-ATPase activity of these reconstituted membranes became appreciable above 20 degrees C and 30 degrees 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 degrees C and 39 degrees 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 dimyristoylphosphatidylcholine-recombinant, measured in terms of hydrogen-deuterium exchange reaction kinetics, showed a jump at about 27 degrees 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 al. (1978) J. Biol. Chem. 253, 6879-6887).
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013816:Thermodynamics
D008563:Membrane Lipids; D003903:Deuterium; D000252:Calcium-Transporting ATPases