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

Abstract

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 (K_d 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.