Restoration of Ca2+-transport in sarcoplasmic reticulum ATPase solubilized with octaethyleneglycol mono n-dodecyl ether.
One mg protein/ml of sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were solubilized with 50 mg/ml of octaethyleneglycol mono n-dodecyl ether (C12E8) in a solution containing 5 mM CaCl2, 0.1 M KCl, and 20% glycerol at pH 7.5. When 30 mg/ml of soybean lecithin was added to this mixture and then incubated with Bio-beads SM-2 at 20 degrees C for 1.5 h to remove the detergent from the mixture, proteoliposomes were formed. This process restored Ca2+-uptake activity to approximately 50% of that of control sR. However, Ca2+-transport was not observed when SR membranes were formed without the addition of soybean lecithin. The reconstituted vesicles also catalyze Ca2+-release, which is coupled to the backward reaction which forms ATP from ADP and P1 in the presence of a Ca2+-gradient across the membrane. When the reconstituted vesicles were subjected to equilibrium centrifugation in a 5 to 25% glycerol density gradient, all of the Ca2+-transport activity was closely associated with the fraction containing soybean liposome.
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000494:pharmacology*; D066298:In Vitro Techniques; D010713:Phosphatidylcholines / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*; D013997:Time Factors
D003902:Detergents; D010713:Phosphatidylcholines; D013501:Surface-Active Agents; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000251:Adenosine Triphosphatases; D002118:Calcium