Quantitative analysis of cell surface-associated SV40 large T antigen using a newly developed 3H-protein A binding assay.
We have established a sensitive assay for the quantitative determination of large T antigen determinants on the surface of living simian virus 40 (SV40)-transformed cells (mKSA). Cells in suspension culture were incubated with monoclonal antibodies specific for large T antigen (KT3, directed against the carboxyterminus of large T antigen, and PAb 108, directed against an aminoterminal determinant on large T antigen). After incubation with secondary antibody (rabbit anti-mouse IgG), followed by incubation with 3H-protein A, the cells were sequentially extracted first with the nonionic detergent NP-40, followed by ultrasonication and extraction with the zwitterionic detergent Empigen BB. NP-40 solubilized large T antigen associated with NP-40-soluble constituents of the plasma membrane, whereas Empigen BB solubilized the plasma membrane lamina-associated subclass of large T antigen (U. Klockmann and W. Deppert, 1983, EMBO J., 7, 1151-1157). The amount of cell surface-bound 3H-protein A in the NP-40 and Empigen BB extracts was determined by liquid scintillation counting. In agreement with earlier reports, cell surface large T antigen was mainly found in association with the plasma membrane lamina (PML). Since the specific activity of 3H-protein A was known, it was possible to calculate the number of surface-bound 3H-protein A molecules, and thus to estimate the average number of surface-exposed amino- and carboxyterminal determinants of large T antigen per cell. KT3 recognized about 450-900 carboxyterminal determinants, while PAb 108 bound to about 1200-2400 aminoterminal determinants on the surface of a single mKSA cell. The cellular protein p53 also was detected on the surface of mKSA cells and was found to be present in amounts comparable to cell surface large T antigen.
Rinke
Y
Y
Abteilung Biochemie, Universität Ulm, Federal Republic of Germany.
Deppert
W
W
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis; D001667:Binding, Competitive; D002461:Cell Line, Transformed; D000939:Epitopes / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis; D011233:Precipitin Tests; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D000954:Antigens, Surface; D000939:Epitopes; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D013205:Staphylococcal Protein A; D016159:Tumor Suppressor Protein p53