Analysis of the Tumor Necrosis Factor (TNF) Receptor of Various Tumor Cells

The existence of TNF receptors on TNF sensitive tumor cells was elucidated by specific binding assay using radioiodinated human recombinant TNF （125I-TNF）. A close correlation （r=0.855） was shown between the receptor number and the susceptibility of tumor cells against TNF. The cytotoxic activity of TNF was quenched by anti TNF monoclonal antibody （IV3-E）, which inhibits the specific binding of TNF to its receptor, indicating that the formation of TNF-receptor complex is a required process for its cytocidal action. We further extended our investigation to identify the TNF receptor on the KYM cell membrane by cross-linking the 125I-TNF with the presumed receptor site using disuccinimidyl suberate （DSS）. Four radioactive bands with a molecular weight of 145K, 50K, 35K and 17K daltons were observed on SDS-PAGE. The latter three bands corresponded to the trimer, dimer, monomer of TNF, respectively. The 145K dalton band represented TNF-receptor complex because it was eliminated by the addition of excess cold TNF or IV3-E antibody. A radioactive peak with 145K dalton was also confirmed by gel filtration chromatography. Since the molecular size of native TNF as estimated by gel filtration was 50K dalton consisting of three identical peptides, the molecular weight of the TNF receptor itself was calculated to be 95K dalton.departmental bulletin pape