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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA038773-06
Application #
3177038
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1984-05-01
Project End
1991-04-30
Budget Start
1989-05-01
Budget End
1990-04-30
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Type
Schools of Arts and Sciences
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Nair, S; Dey, R; Sanford, J P et al. (1992) Molecular cloning and analysis of an eIF-4A-related rat liver nuclear protein. J Biol Chem 267:12928-35
Yilla, M; Doyle, D; Sawyer, J T (1992) Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system. J Cell Biol 118:245-52
Sanford, J P; Eddy, R L; Doyle, D et al. (1991) Assignment of human asialoglycoprotein receptor gene (ASGR1) to chromosome 17p11-13. Genomics 11:779-81
Sawyer, J T; Doyle, D (1990) Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation. Proc Natl Acad Sci U S A 87:4854-8
Petell, J K; Quaroni, A; Hong, W J et al. (1990) Alteration in the regulation of plasma membrane glycoproteins of the hepatocyte during ontogeny. Exp Cell Res 187:299-308
Sanford, J P; Doyle, D (1990) Mouse asialoglycoprotein receptor cDNA sequence: conservation of receptor genes during mammalian evolution. Biochim Biophys Acta 1087:259-61
Hong, W J; Doyle, D (1990) Molecular dissection of the NH2-terminal signal/anchor sequence of rat dipeptidyl peptidase IV. J Cell Biol 111:323-8
Hong, W J; Petell, J K; Swank, D et al. (1989) Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels. Exp Cell Res 182:256-66
Hong, W J; Piazza, G A; Hixson, D C et al. (1989) Expression of enzymatically active rat dipeptidyl peptidase IV in Chinese hamster ovary cells after transfection. Biochemistry 28:8474-9
Hong, W J; Doyle, D (1989) Cloning and analysis of cDNA clones for rat kidney alpha-spectrin. J Biol Chem 264:12758-64

Showing the most recent 10 out of 20 publications