Examination of transcellular membrane protein polarity of bovine aortic endothelial cells in vitro using the cationic colloidal silica microbead membrane-isolation procedure.
In this report we describe a rapid, high-yield protocol for the isolation of apical (AP) and basolateral (BL) plasma membrane domains from monolayers of bovine aortic endothelial cells (BAECs) grown on tissue culture dishes as well as microcarrier beads. Using a modified cationic colloidal silica microbead membrane-isolation procedure, which deposits a uniform silica-polyacrylate pellicle over the entire AP membrane surface, a 4- to 9.6-fold relative enrichment of AP membrane and a 3.55- to 3.67-fold relative enrichment of BL membrane was obtained when the isolated domains were examined for silica and Na+/K(+)-ATPase, respectively. Immunoblotting of the isolated membrane domains displayed the presence of angiotensin-converting enzyme (ACE) exclusively in the AP domain and collagen receptors (CRs) highly enriched in the BL membrane domain when monolayers were grown on a gelatin substratum.
Stolz
D B
DB
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003.
Jacobson
B S
BS
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D012822:Silicon Dioxide
D008565:Membrane Proteins; D012822:Silicon Dioxide