[23] Reconstitution of skeletal muscle sarcoplasmic reticulum membranes: Strategies for varying the lipid/protein ratio

Publisher Summary This chapter describes reconstitution methodology for the preparation of functionally active reconstituted sarcoplasmic reticulum (SR) membrane vesicles with high protein content comparable to that in the original SR. The nature of the dissociation and reconstitution process for SR using deoxycholate has recently been elucidated. Titration of SR with DOC first involves the selective extraction of bilayer phospholipid to yield a "limit membrane" consisting of 48 mol of phospholipid per mole of CPP (compared with 115 for the original SR membrane), and second, the solubilization of the CPP and associated phospholipid from the limit membrane. Membrane assembly is essentially the reversal of dissociation. Raising the temperature results in reformation of a limit membrane. As detergent is removed, phospholipid gradually adds to the limit membrane, the phospholipid content increases, and vesicles are formed. Dissociation is carried out in the cold at 0–4°. A suspension of SR vesicles at 6 mg protein/ml in dissociation buffer is used. This is prepared by diluting the SR to 12 mg protein/ml with solution B, and mixing with an equal volume of solution A sucrose. Reconstitution of SR vesicles from solubilized SR occurs optimally at room temperature, whereas little or no Ca 2÷ transport activity is recovered when the procedure is carried out at 0°.