Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes

Abstract

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12–16-fold for angiotensin-converting enzyme activity and 8–10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na + + K +)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC 6 and XVD 2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD 2 reacted with an antigen, indicating that the antibody XIVC 6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD 2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets. These monoclonal antibodies will be useful for monitoring the separation of luminal and abluminal membrane domains and in the immunoaffinity purification of these domains.