title: Bioactive human recombinant tumor necrosis factor-alpha: an unstable dimer?
abstract: As determined by native polyacrylamide gel electrophoresis (PAGE) and gel chromatography the molecular mass of native tumor necrosis factor (TNF)-alpha was approximately 35 kDa. When incubated at low concentrations (less than 1 nM) 125I-labeled TNF-alpha and unlabeled TNF-alpha rapidly multimerized or dissociated into monomers and bioactivity decreased. Sodium dodecyl sulfate (SDS)-PAGE analysis of cross-linked 125I-labeled TNF-alpha demonstrated bands of multi- and trimeric TNF-alpha in addition to dominating bands of dimers and monomers. Tri-, di- and monomeric TNF-alpha were recovered from SDS-PAGE gels and allowed to renature. Of the original receptor-binding activity, 10%-15% was obtained with cross-linked TNF-alpha dimers, whereas none was recovered from preparations of trimeric TNF-alpha. Multimeric and monomeric TNF-alpha exhibited little or no binding activity, and cell-bound, cross-linked TNF-alpha which was dissociated from cellular binding sites was mainly dimeric. 125I-labeled TNF-alpha 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 x 10(3). Cross-linking of TNF-alpha to binding sites in U-937 and LAK cells yielded a receptor-ligand complex of about 80/90 kDa. At 37 degrees C, 125I-labeled TNF-alpha 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-alpha-mediated cytolysis of L-929 cells and caused a quantitatively corresponding reduction in cellular TNF-alpha uptake, indicating that L-929 lysis was mediated by receptors.
authors.affiliation: Department of Clinical Immunology, Skejby Sygehus, Aarhus N, Denmark.
authors.forename: C M
authors.initials: CM
authors.lastname: Petersen
authors.forename: A
authors.initials: A
authors.lastname: Nykjaer
authors.forename: B S
authors.initials: BS
authors.lastname: Christiansen
authors.forename: L
authors.initials: L
authors.lastname: Heickendorff
authors.forename: S C
authors.initials: SC
authors.lastname: Mogensen
authors.forename: B
authors.initials: B
authors.lastname: Møller
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015979:Killer Cells, Lymphokine-Activated / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D008744:Methylamines; D011108:Polymers; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C027451:methylamine