Use of a density modification technique for isolation of the plasma membrane of rod outer segments

Abstract

The external surface of rod outer segments contains receptors for the Jack Bean lectin, concanavalin A. Using isolated intact bovine rod outer segments, we have modified the density of the outer segment plasma membranes by means of polystyrene beads carrying covalently linked concanavalin A. After hypotonic lysis the bulk of the disk membranes can be removed and a plasma membrane fraction is isolated. The plasma membrane preparation contains 1.5% of total outer segment rhodopsin and 2.7% of total outer segment protein. It shows very little contamination with inner segment plasma membrane. Contamination with disk membranes appears to be low as well. Fatty acid analysis reveals that the plasma membranes are more saturated than the highly unsaturated disk membranes. Gel electrophoresis shows the presence of at least six additional polypeptides besides rhodopsin.