9] Isolation of canine cardiac sarcoplasmic reticulum

Abstract

Publisher Summary This chapter focuses on the isolation of canine cardiac sarcoplasmic reticulum. The sarcoplasmic reticulum (SR) is a specialized intracellular membrane system in muscle that regulates the myoplasmic calcium (Ca 2+ ) level—thereby, controlling contraction and relaxation. In cardiac muscle, the SR network is less extensive than it is in skeletal muscle, and cell surface membranes (sarcolemma and transverse tubule) constitute much of the total membrane content. The isolation procedure described in the chapter yields canine cardiac SR of high purity, which exhibits efficient, high specific activity Ca 2+ transport, and which is stable to multiple freezings and thawing, and to long-term cryogenic storage. These attributes have made possible the following advances: The determination of the oligomeric structure of the Ca 2+ -pump protein in cardiac SR membranes using radiation inactivation analysis and the reconstitution of the Ca 2+ -pumping function of cardiac SR. Cardiac SR isolated by this procedure includes both ryanodine-sensitive and ryanodine-insensitive subfractions, and can serve as starting material for preparing SR subfractions by loading with Ca 2+ oxalate or phosphate, using modifications of published procedures.