Oligomeric tumour necrosis factor alpha slowly converts into inactive forms at bioactive levels.
The stability of oligomeric human tumour necrasis factor alpha (TNF) at bioactive levels has been studied by two immunoenzymatic assays: one able to specifically detect oligomeric and not monomeric TNF (O-e.l.i.s.a.) and the other able to detect both forms (OM-e.l.i.s.a.). The selectivity of O-e.l.i.s.a. and OM-e.l.i.s.a. for oligomeric and monomeric TNF was demonstrated with isolated forms prepared by partial dissociation of recombinant TNF with 10% (v/v) dimethyl sulphoxide and gel-filtration h.p.l.c. Evidence for instability of oligomeric TNF were obtained in physiological buffers, as well as in serum and cell-culture supernatants, as a function of TNF concentration. In particular, only a half of the TNF antigen was recovered in the oligomeric form after 72 h incubation (37 degrees C) at 0.12 nM, whereas no apparent dissociation was detected at 4 nM. The structural changes observed at picomolar concentrations were rapidly reversed by raising the concentration of TNF to about 2 nM by ultrafiltration, suggesting that subunit dissociation and reassociation reactions occur in the picomolar and nanomolar range respectively. The cytolytic activity of L-M cells correlates with oligomeric-TNF levels after incubation at picomolar concentrations. Moreover, isolated oligomeric TNF was cytotoxic towards L-M cells, whereas monomeric TNF was virtually inactive. In conclusion, the results suggest that bioactive oligomeric TNF is unstable at picomolar levels and slowly converts into inactive monomers, supporting the hypothesis that quaternary-structure changes in TNF may contribute to the fine regulation of TNF cytotoxicity.
Corti
A
A
Molecular Immunology and Biochemistry Unit, TECNOGEN SCpA, Milan, Italy.
Fassina
G
G
Marcucci
F
F
Barbanti
E
E
Cassani
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D002470:Cell Survival / Q000187:drug effects; D004121:Dimethyl Sulfoxide; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D004121:Dimethyl Sulfoxide