Bioactive human recombinant tumor necrosis factor-α: an unstable dimer?

As determined by native polyacrylamide gel electrophoresis (PAGE) and gel chromatography the molecular mass of native tumor necrosis factor (TNF)-α was approximately 35 kDa. When incubated at low concentrations (< 1 nM) 125I-labeled TNF-α and unlabeled TNF-α rapidly multimerized or dissociated into monomers and bioactivity decreased. Sodium dodecyl sulfate (SDS)-PAGE analysis of cross-linked 125I-labeled TNF-α demonstrated bands of multi- and trimeric TNF-α in addition to dominating bands of dimers and monomers. Tri-, di- and monomeric TNF-α were recovered from SDS-PAGE gels and allowed to renature. Of the original receptor-binding activity, 10%–15% was obtained with cross-linked TNF-α dimers, whereas none was recovered from preparations of trimeric TNF-α. Multimeric and monomeric TNF-α exhibited little or no binding activity, and cell-bound, cross-linked TNF-α which was dissociated from cellular binding sites was mainly dimeric. 125I-labeled TNF-α bound to lymphokine-activated killer (LAK) cells and binding kinetics were much similar (Kd approximately 100 pM) to those reported in other normal cell types. The number of receptors per LAK cell was approximately 4 × 103. Cross-linking of TNF-α to binding sites in U-937 and LAK cells yielded a receptor-ligand complex of about 80/90 kDa. At 37 °C, 125I-labeled TNF-α was rapidly internalized and degraded in L-929, U-937 and LAK cells. Degradation of ligand and recycling of receptors were blocked in the presence of methylamine. Methylamine significantly inhibited TNF-α-mediated cytolysis of L-929 cells and caused a quantitatively corresponding reduction in cellular TNF-α uptake, indicating that L-929 lysis was mediated by receptors.