Our previous investigations of glycoproteins isolated from the plasma membranes of normal and neoplastic rat livers revealed many qualitative and quantitative differences when analyzed by two- dimensional polyacrylamide gel electrophoresis (2D-PAGE). The main goal of this project is to purify and characterize the specific glycoproteins whose expression is markedly altered during chemically induced hepatocarcinogenesis in order to understand their role either as markers or causal agents during cell transformation. Results obtained are as follows: The previous isolation procedure was modified to more efficiently purify the glycoproteins of interest. The new method utilizes modified conditions for the Concanavalin-A (Con A) affinity chromatography and fast protein liquid chromatography (FPLC) using a Superose-12 column. This procedure provides a fraction that is enriched in the various glycoproteins of interest and enabled the final purification to be achieved by a single 2D-PAGE experiment. These separated components were transferred to an Immobilon-P membrane by electroblotting. It was necessary to optimize the conditions of the 2D-PAGE and electroblotting for these specific glycoproteins. In addition, the best procedure for amino acid sequencing (gas phase) from these membranes was established. Using these techniques, it was possible to determine the N-terminal amino acid sequence for 4 of the 9 glycoproteins analyzed. The remaining 5 components of interest were not sequencable in this manner, presumably because of blocked N-termini. A peptide comprised of the first 13 N-terminal residues of the fragment RLMP-I was synthesized, purified, and used to produce rabbit antibodies for use in the larger scale purification of the whole protein and in various biological studies. The glycoproteins from normal and transformed rat liver epithelial (RLE) cells are currently being characterized by the 2D-PAGE system.