Isolation and characterization of the apical surface of polarized Madin-Darby canine kidney epithelial cells.
Academic Article
Overview
abstract
We have developed a fast and efficient method to isolate the apical surface of Madin-Darby canine kidney epithelial cells. After confluent cell monolayers were coated with alternate layers of cationized colloidal silica and a polyanion, 60% of the apical surface was recovered as large membrane sheets through the application of a polylysine-coated glass surface. Scanning electron microscopy of the cytoplasmic aspect of the apical surface revealed a honeycomb pattern given by the cell borders fractured at or above the level of the tight junctions. By transmission electron microscopy, the apical preparation appeared to be composed of plasma membrane and a thin layer of cytoplasm. Enzyme assays and immunoblots demonstrated a 6- to 7-fold enrichment of an apical marker and a low level of contamination by cytoplasmic and basolateral markers. After removal of cytosolic contaminants and peripheral membrane proteins by alkaline extraction, apical integral membrane proteins were characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Lectin blotting and [3H]glucosamine labeling identified two major sialoglycoproteins of apparent Mr 200,000 and 100,000. The apical membrane sheets here described provide a useful model for systematic characterization of the molecular components of the membrane, for reconstitution of lipid and protein transport in cell-free systems, and for study of the interactions of submembranous cytoskeletal proteins with the apical plasma membrane domain.
