BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Reconstitution of Sarcoplasmic Reticulum-$Ca^{2+}$ Release Channels into Phospholipid Vesicles : Investigation of Conditions for Functional Reconstitution

  • Yang, In-Sik (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Lee, Hee-Bong (Department of Biochemistry, College of Natural Sciences, Kangwon National University)
  • Received : 1994.10.04
  • Published : 1995.03.31
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Abstract

The ryanodine-receptor $Ca^{2+}$ release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified $Ca^{2+}$ release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.

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