The overall objective of the proposed research is to identify by a variety of methods, including external labeling, immunological, cell fractionation and cytochemical the proteins and the glycoproteins that are externally oriented in the plasma membrane of hepatoma cells and primary cultures of rat hepatocytes. Lactoperoxidase catalyzed iodination and tritiated sodium borohydride reduction of cells in situ are used to label externally accessible proteins and glycoproteins. These proteins then are resolved by two-dimensional polyacrylamide gel electrophoresis in the presence of detergents. A number of these proteins are being purified and antibodies both conventional and monoclonal are being prepared to them. Cell fractionation in combination with immunocytochemical localization studies at both the light and electron microscopic level then are used to assign each of the proteins to the domain of the plasma membrane in which it resides. Antibodies to different proteins in different domains of the hepatocyte plasma membrane next are used to isolate the different domains of the membrane in pure form using novel methodology not based on ultracentrifugation. The composition of the other proteins in the purified domain is being established by two-dimensional polyacrylamide gel electrophoretic analyses. The turnover behavior of each of the proteins in each of the domains is being assessed utilizing the dual isotopic labeling procedures that have already been developed in our laboratory. The time course of appearance during development of domain specific proteins also is being examined. The goal of these studies is to establish if the domain is the unit for membrane turnover in this complex cell type and to determine which of the three most plausible mechanisms; lysosomal fusion, nonlysosomal proteases and/or shedding, is involved in the regulation of plasma membrane protein concentration in hepatocytes. As a corollary to this primary goal studies are being conducted to follow the pathway of biogenesis of specific proteins to their domain of residence in both the plasma membrane of the differentiated hepatocyte and the hepatocyte during development.
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