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Subcellular Localization of Simian Virus 40 Large Tumor Antigen

Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [³⁵S]-methionine- or ³²P_i-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of 96,000 was detected in both subcellular fractions. Minor components of 68,000 and 56,000 with anti-T reactivity which labeled with [³⁵S]methionine were also detected in both fractions from H-50 cells, as were components of 140,000 and 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in ³²P_i-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [³H]thymidine labeling, NADH-diaphorase activity, and Na⁺-K⁺-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.

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