[45] Biosynthesis of sarcoplasmic reticulum proteins

Publisher Summary This chapter describes methods for in vivo and in vitro synthetic studies of biosynthesis of sarcoplasmic reticulum proteins. In cell culture, the initiation of synthesis of the Ca 2+ , Mg 2+ -ATPase and the intrinsic glycoprotein occurs at about the time of myoblast fusion. Calsequestrin and the ATPase are synthesized on membrane-bound polyribosomes—calsequestrin with a signal sequence, and the ATPase without one. Initiation of synthesis of the extrinsic protein, calsequestrin, and the high-affinity Ca 2+ binding protein occurs several hours earlier, well before fusion begins. The chapter also discusses immunoprecipitation, isolation of RNA, and translation system for muscle mRNA. Calsequestrin can also be purified from the dialyzed, fractionated extracts by two-dimensional gel electrophoresis. The preparation of rabbit reticulocyte lysate is suitable for translation of rabbit muscle mRNA.