The long-term objective of this research is to understand how epithelial cells establish and maintain their polarized surface. Six new rat hepatocyte plasma membrane sialo-glycoproteins have been identified and localized both morphologically (ie, immunofluorescence and immuno-electron microscopy) and biochemically (gradient separation of plsma membrane vesicles and immunochemical analysis). Five are domain-specific: there are restricted to the bile canalicular domain and two to the sinusoidal/lateral domain. One is present in all domains. Further limited characterization of these molecules will include: carbohydrate compositions using lectin, enzymatic and chemical methods; the transmembrane character of selected antigens using controlled proteolysis on membranes vesicles of different orientations; and microdomain distributions on isolated membranes and tissue using immuno-electron microscopy. Steady state parameters of the membrane glycoproteins (ie, cellular content, half-lives, intracellular distributions) will be measured in vivo and in the perfused liver using immunochemical methods in combination with subcellular fractionation. Kinetic experiments will be conducted in order to trace the intracellular biogenetic, degradative and recycling pathways of these cell surface glycoproteins. Different labeling protocols (ie, metabolic labels (amino acids, monosaccharides), exogenous reagents (125I-lactoperoxidase, trinitrobenzene sulfonate)) will be used in the isolated, perfused liver, followed by analytical subcellular fractionation at various times, specific immunoprecipitation, and analysis of the rates at which the labeled antigens are found in various compartments. Vesicular carriers of the antigens at key (sorting) points in these various pathways will be identified, isolated by immunoadsorption using antibodies directed against cytoplasmic domains of proven transmembrane proteins and characterized biochemically, morphologically and immunologically. Next, the domain-specific glycoproteins will be studied in isolated hepatocytes where cell polarity has been perturbed. Liver will be enzymatically dissociated and the amounts and surface distribution of these antigens first assessed on single cells using immunochemical and immuno-morphological techniques. Then the effects of culture conditions (eg, substratum, extent of initial dissociation, etc.) on the distributions and dynamics (biogenesis, degradation, and recycling) of these molecules will be examined using a kinetic approach and analytical fractionation.
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