The overall objective of this research is to identify, by external labeling methods, 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 as the methods to label externally accessible proteins and glycoproteins. Cell fractionation in combination with immunocytochemical localization studies at both the light and electron microscopic level are then used to assign each of the proteins to a domain of the plasma membrane in which it resides. Antibodies to different proteins in different domains of the hepatocyte plasma membrane are then being used to isolate the different domains of the membrane in pure form. The protein composition of the purified domain will then be established by two-dimensional, polyacrylamide gel electrophoretic analyses. The turnover behavior of each of the proteins in each of the domains will then be assessed utilizing the dual isotopic labeling procedures that have already been developed in our laboratory. 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. (A)
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