Fractionation and reconstitution of the sarcoplasmic reticulum Ca2+ pump solubilized and stabilized by CHAPS/lipid micelles.
A procedure for solubilization, fractionation, and reconstitution of sarcoplasmic reticulum (SR) protein is presented. The SR protein is solubilized with the zwitterionic detergent CHAPS in the presence of added 5-mM phosphatidylcholine and 20% glycerol, which stabilize the reconstitutable Ca2+ transport activity. For reconstitution the solubilized SR protein is incorporated into preformed French-pressed unilamellar vesicles that had been treated with 10-mM sodium cholate. By passing the proteoliposomes through a centrifuged Sephadex G-50 column that had been equilibrated with potassium oxalate, the detergent is removed, and the proteoliposomes become sealed with potassium oxalate trapped inside. This procedure requires less than 2 h and results in Ca2+ uptake active of over 1 mumol/min/mg of protein. The solubilized SR protein was fractionated on a DEAE-Biogel A column. A fraction containing the Ca2+-ATPase but not the Mr 55,000 glycoprotein had reconstitutable Ca2+ uptake activity of 2.2 mumol/min/mg of protein. Inclusion of the Mr 55,000 glycoprotein during the reconstitution procedure did not increase the Ca2+ uptake activity of the reconstituted fraction containing the Ca2+-ATPase. This result demonstrates that the glycoprotein is not required for Ca2+ uptake.
D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification* / Q000378:metabolism; D002793:Cholic Acids; D008081:Liposomes / Q000378:metabolism*; D008970:Molecular Weight; D010070:Oxalates; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D002793:Cholic Acids; D008081:Liposomes; D010070:Oxalates; D000252:Calcium-Transporting ATPases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium