Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity
✍ Scribed by Farida Nato; Roland Bourrillon
- Publisher
- John Wiley and Sons
- Year
- 1982
- Tongue
- English
- Weight
- 844 KB
- Volume
- 18
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
A major cell surface sialoglycoprotein with, Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells.
The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaI04 followed by reduction with NaB3H4. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP 112, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,OOO daltons.
it could be labeled metabolically with D-(3 H) glucosamine and externally through the nonpenetrating periodate-NaB3H4 system. concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP 112 could be an integral protein.
N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both 0-and N-linked to the polypeptide chain, most of them being 0-linked.
Finally, GP 112 has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.
The location of GP 112 on the cell surface was confirmed by the fact that GP 112 could not be removed from the cell surface by high salt Analysis of the carbohydrate composition of GP 112 revealed galactose,