Use of a sensitive receptor binding assay to discriminate between full-length and truncated human recombinant tumor necrosis factor proteins.
A radioreceptor assay (RRA) capable of detecting picomolar concentrations of human recombinant tumor necrosis factor (TNF) was used to compare the relative binding affinities of genetically engineered full-length and truncated TNF proteins. The specific cell-surface receptors for TNF present on the human cervical carcinoma cell line ME-180 were characterized as having a Kd of 0.2 nM and a density of 2700 sites/cell. Conditions were then defined for an RRA that maximized the specific binding of 125I-TNF to this adherent cell line. Incubation of ME-180 cells with 125I-TNF at 37 degrees C in the presence of 0.02% sodium azide resulted in a 4-fold increase in assay sensitivity and a doubling of specific counts bound, as compared to binding done at 4 degrees C with or without sodium azide. Inhibition of receptor-ligand internalization under these conditions was a likely reason for the increases. This system was utilized to compare low concentrations of the full-length TNF protein and a genetically altered TNF protein (mutein) which lacks the 10 N-terminal amino acids and contains an N-terminal methionine. Previous studies showing the truncated TNF to be 2- to 3-fold lower in cytotoxic activity on a variety of tumor cell lines were corroborated by our findings that the mutein was also three and one-half times lower in relative affinity for the TNF receptor on ME-180 cells. These results suggest a possible role for these residues in receptor binding and illustrate the use of a highly sensitive RRA for the evaluation of TNF molecules altered by recombinant DNA technology.
D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000378:metabolism*; D019810:Sodium Azide; D014409:Tumor Necrosis Factor-alpha; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*
D001386:Azides; D006023:Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019810:Sodium Azide