Biological epithelia which separate physiological compartments act as permeability barriers to the transepithelial passage of ions and molecules. The spaces between the cells, called the paracellular pathway, are sealed by a gasket-like intercellular junction, the zonula occludens (ZO). The ZO is the apical-most member of a series of intercellular junctions, collectively termed the junctional complex. A novel preparation of isolated junctional complexes from rodent liver has prepared, which is demonstrated by morphological techniques to contain structural components of the ZO. Polyacrylamide gel analysis of these complexes reveals a greatly simplified profile of polypeptides, compared to the whole hepatocyte plasma membrane. Using monoclonal and polyclonal antibody approaches, it is proposed in this application to generate specific antisera to these junctional complex preparations and to identify the specificity of these antisera by immunoreplica techniques. Using immunoelectron microscopy, the anatomical locations of the antigens recognized by these antisera will be mapped within the junctional complex, with the goal of specifically identifying the molecular components of the ZO. Once identified, the biogenesis and assembly of the ZO will be studied using immune precipitation of antigens following synthesis in either whole liver or rough microsome enriched fractions of polyA mRNA. Hopefully, these antisera will also prove valuable in studies of biological transport in cultures of transporting epithelia.
