Selective release of integral proteins from human erythrocyte membranes by hydrostatic pressure

Abstract

The overt effect of pressure on biological membranes is mediated predominantly through lipid condensation and disintegration of cytoskeletal polymers. These may lead to selective shedding of integral proteins, which could then be isolated by conventional means. In this study we have used the well characterised human erythrocyte membrane in order to establish the technical requirements for future use of pressure, as an alternative to detergents, in isolation of membrane proteins. Pressure of varying magnitude (300–1640 bar) and duration (5–60 min) was applied on human erythrocyte ghost membranes in suspension at different temperatures (4, 24 and 37°C) and in the presence of various solutes. After ultracentrifugation protein and lipids remaining in the supernatant were quantified and analysed. It is indicated that selective integral membrane proteins can be shed off under defined conditions and presumably remain in solution by the support of strongly associated phospholipids and specific solutes. On the basis of our findings a series of technical recommendations for the isolation of specific membrane proteins is outlined.