Identification and partial characterization of the major galactoproteins present at the surface of AS-30D hepatocellular carcinoma cells.

The major plasma membrane glycoproteins of AS-300 hep atoceblular carcinoma cells were radiolabebed by one or more cell surface-specific methods: D-galactose oxidase-NaB3H4; NalO4-NaB3H4;or bactoperoxidase-catalyzed iodination. The major radlobabeledgalactoproteins 1a, 1b, and 2 were resolved by affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin lband two-dimensional gel electrophore sis. These gabactoproteins had apparent molecular weights in the range of 110,000 to 160,000 as judged by their behavior in sodium dodecyb sulfate-polyacrylamide gel ebectrophoresis in 7.5% acrylamide gels. Each of the gabactoprotemnsbound to Sepharose-conjugated wheat germ agglutinin and exhibited charge heterogeneity. Gabactoprotein 1a, a component sebec tively labeled by D-galactose oxidase-NaB3H4 or Na104NaB3H4,was further characterized. Sodium dodecyl sulfate polyacrybamidegel electrophoresis of galactoprotein 1a in gels of varying acrylamide concentration allowed approximation of its subunit molecular weight (128,000) and suggested a high carbohydrate content. Treatment of cells with neuraminidase prior to radiolabeling was accompanied by a shift in the isoe bectric points of the heterogeneous species of galactoprotemn 1a to more basic pH's, indicating that variation in siabic acid content was at least partially responsible for its charge heter ogeneity. When cells were pretreated with high concentrations of neuraminidase, labeling of gabactoprotemn1a by the Na104NaB3H4 method was reduced only 50%, suggesting that a significant fraction of the sialic acid is resistant to cleavage by neuraminidase. Exposure of cells labeled by the NalO4-NaB3H4 method to exogenous papain resulted in degradation of gal actoprotein 1a and the appearance of a new radiolabebed glycopeptide having an apparent molecular weight of 63,000 in 7.5% acrylamide gels.