Abstract
The major plasma membrane glycoproteins of AS-30D hepatocellular carcinoma cells were radiolabeled by one or more cell surface-specific methods: d-galactose oxidase-NaB3H4; NalO4-NaB3H4; or lactoperoxidase-catalyzed iodination. The major radiolabeled galactoproteins 1a, 1b, and 2 were resolved by affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin II and two-dimensional gel electrophoresis. These galactoproteins had apparent molecular weights in the range of 110,000 to 160,000 as judged by their behavior in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 7.5% acrylamide gels. Each of the galactoproteins bound to Sepharose-conjugated wheat germ agglutinin and exhibited charge heterogeneity. Galactoprotein 1a, a component selectively labeled by d-galactose oxidase-NaB3H4 or NalO4-NaB3H4, was further characterized. Sodium dodecyl sulfate-polyacrylamide gel 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 isoelectric points of the heterogeneous species of galactoprotein 1a to more basic pH's, indicating that variation in sialic acid content was at least partially responsible for its charge heterogeneity. When cells were pretreated with high concentrations of neuraminidase, labeling of galactoprotein 1a by the NalO4-NaB3H4 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 galactoprotein 1a and the appearance of a new radiolabeled glycopeptide having an apparent molecular weight of 63,000 in 7.5% acrylamide gels.
Footnotes
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↵1 Supported by grants from the National Cancer Institute (CA 18829 and CA 27377), the Paul and Mary Haas Foundation, and the George and Josephine Hamman Foundation.
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↵2 Recipient of predoctoral fellowships from the Rosalie B. Hite Foundation and the J. S. Abercrombie Foundation.
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↵3 To whom requests for reprints should be addressed.
- Received December 11, 1979.
- Accepted May 8, 1980.
- ©1980 American Association for Cancer Research.