Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells.
Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10(-10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein.
D002460:Cell Line; D003432:Cross-Linking Reagents; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha
D003432:Cross-Linking Reagents; D006023:Glycoproteins; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide