Alzani R, Cozzi E, Corti A, Temponi M, Trizio D, Gigli M, Rizzo V
Dibit, San Raffaele H Scientific Institute, Milan, Italy.
Biochemistry. 1995 May 16;34(19):6344-50. doi: 10.1021/bi00019a012.
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.
采用尺寸排阻色谱法并结合γ计数器进行离线监测，对添加了¹²⁵I标记形式的人肿瘤坏死因子α（TNF）的解聚过程进行了定量研究。在不存在或存在不同量（10、100、1000 μM）苏拉明（一种体外TNF生物活性抑制剂，可促进TNF解聚）的情况下，对TNF的寡聚状态随其自身浓度（以亚基计为0.3 - 7500 nM，相对分子质量为17,000）的变化进行了详细研究。假设每一步的解离常数相同（Kd1 = 0.2 nM），用连续解离过程（三聚体→二聚体→单体）对三聚体形式含量与总TNF浓度的关系进行建模。尽管通常无法获得二聚体的色谱分离，但该模型是用于数据拟合的最简单模型。包含TNF三聚体构象转变为更易解聚状态（Kd2 = 400 nM）的模型能够实现所有数据的最佳拟合，而苏拉明结合有利于这种转变。用相同方法对TNF解离进行动力学研究，得出三聚体的解聚速率值：在4至20℃之间，平均koff约为4×10⁻⁵ s⁻¹（t1/2约为5小时），对苏拉明浓度的依赖性较小；在37℃时，存在1 mM苏拉明时解聚速率显著增加（koff = 2.3×10⁻⁴ s⁻¹，t1/2 = 0.8小时）。在比上述三聚体半衰期短得多的孵育时间后获得的苏拉明对TNF受体结合抑制的数据表明，苏拉明与TNF三聚体的结合是受体结合抑制的早期机制。
