ISOLATION OF HEPATOCYTE PLASMA MEMBRANE PROTEINS FROM NORMAL & NEOPLASTIC CELLS

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Our previous investigations of glycoproteins isolated from the plasma membranes of normal and neoplastic rat livers using two- dimensional gel electrophoresis (2D-PAGE) have revealed many differences, both qualitative and quantitative. The main goal of this project is to isolate, purify, and structurally 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 in the process of cell transformation. Results obtained so far are as follows: Attempts have been made to isolate a specific glycoprotein (molecular weight, 200 KD; pI 5.8), which is downregulated during cell transformation, from normal rat liver by perfusion, homogenization, ultra-centrifugation, and brief sonication. The glycoprotein was further purified by Concanavalin-A (ConA) affinity chromatography and then gel filtration using a Superose-12 column eluted with Tris buffer containing 6M guanidine. Following desalting by dialysis of the solution against 1.0 M acetic acid, subsequent purification has been achieved by ion-exchange chromatography using a Mono S column eluted with a linear gradient of 1.0 M sodium chloride (NaCl) in ammonium acetate buffer (pH 5.0). Two-dimensional gel electrophoresis has been used to monitor the progress of purification of the glycoprotein at each step.