Mechanism of suramin-induced deoligomerization of tumor necrosis factor alpha.
Deoligomerization of human tumor necrosis factor alpha (TNF), spiked with 125I-labeled form, was studied quantitatively using size-exclusion chromatography and off-line monitoring with a gamma-counter. A detailed investigation of the oligomeric state of TNF was carried out as a function of its own concentration (0.3-7500 nM referred to the subunit, M(r) 17,000) in the absence or in the presence of various amounts (10, 100, 1000 microM) of suramin, an inhibitor of TNF biological activity in vitro, which promotes TNF deoligomerization. The dependence of trimeric form content on total TNF concentration was modeled with a sequential dissociation process (trimer-->dimer-->monomer) assuming an identical dissociation constant for each step, Kd1 = 0.2 nM. This model was used as the simplest for data fitting although, generally, no chromatographic resolution of dimeric species could be obtained. Best fitting of all data could be achieved with a model including a conformational change of TNF trimer into a state more prone to deoligomerization (Kd2 = 400 nM), which was favored by suramin binding. A kinetic study of TNF dissociation by the same method produced values for the deoligomerization rate of trimer: on the average, koff approximately 4 x 10(-5) S-1 (t1/2 approximately 5 h) between 4 and 20 degrees C with little dependence on suramin concentration; at 37 degrees C, a sizable increase is observed in the presence of 1 mM suramin (koff = 2.3 x 10(-4) S-1, t1/2 = 0.8 h). Data of suramin inhibition on TNF receptor binding, as obtained after incubation times much shorter than the above half-life of trimer, indicate that suramin binding to TNF trimer is the early mechanism of receptor binding inhibition.
Alzani
R
R
Dibit, San Raffaele H Scientific Institute, Milan, Italy.
Cozzi
E
E
Corti
A
A
Temponi
M
M
Trizio
D
D
Gigli
M
M
Rizzo
V
V
D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation / Q000187:drug effects; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011994:Recombinant Proteins; D013498:Suramin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
D046911:Macromolecular Substances; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D013498:Suramin