Chapter 11 The (CA2+-Mg2+)-ATPase and other membrane proteins: what reconstitution tells us about the biological membrane

Abstract

Publisher Summary This chapter discusses the reconstitution of the (Ca 2+ –Mg 2+ )-ATPase from sarcoplasmic reticulum. The ATPase is purified as a lipid–protein complex by the solution of the membrane in the detergent, cholate, followed by sucrose gradient centrifugation. A minimum of 30 phospholipid molecules is required to maintain the activity of the ATPase; this number can be compared to the number of phospholipid molecules required to form a complete shell around the ATPase in the membrane. In the lipid–protein complex, the protein is pure, but the phospholipid is a complex mixture representative of that present in the original membrane. The chapter illustrates the technique for reconstituting the ATPase into a phospholipid bilayer of defined composition. The lipid–protein complex is dissolved in cholate and a large excess of the phospholipid of choice is added. It is known that cholate forms disc-like micelles in which the planar cyclopentenophenanthrene rings are apposed and the hydroxyl groups are exposed to the aqueous environment. This may allow the ATPase to dissolve with its phospholipid annulus intact. Purified protein can also be incorporated into pre-formed phospholipid vesicles in the absence of detergent by a freeze/thaw, sonication process.