Fractionation and Reconstitution of the Sarcoplasmic Reticulum Ca²⁺ Pump Solubilized and Stabilized by CHAPS/Lipid Micelles

Abstract

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 μmol/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 M_r 55,000 glycoprotein had reconstitutable Ca²⁺ uptake activity of 2.2 μmol/min/mg of protein. Inclusion of the M_r 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.

Key Words:

• Ca²⁺-ATPase
• liposomes