Isolation of plasma membrane domains from bovine corneal endothelial cells

Abstract

Corneal endothelial cells are polarized cells responsible for pump, barrier and transport functions, which are carried out by proteins located on the apical or basolateral plasma membrane domain. Apical, basolateral, and total plasma membranes of cultured bovine corneal endothelial cells were isolated to characterize their protein constituents. Apical membranes were isolated by applying charged beads onto the cell monolayer, and harvesting the membranes by washing off the beads. Total cell membranes were isolated in cell/bead suspensions followed by sonication and vortexing. Basolateral plasma membranes were scraped off culture dishes after disrupting cells with distilled water. A variety of marker enzyme assays demonstrated, that the plasma membrane domains could be isolated and separated with relative purity, and with only little contamination by intracellular membrane proteins. SDS-polyacrylamide gel electrophoresis showed approximately 50 protein bands for total plasma membranes, 35 protein bands for apical plasma membranes and approximately 45 bands for basolateral plasma membranes.