The active form of tumor necrosis factor is a trimer.
The active form of tumor necrosis factor is a trimer.
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DOI：
10.1016/s0021-9258(18)48183-5
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发表时间：
1987-05
期刊：
影响因子：
--
通讯作者：
R. Smith;C. Baglioni
中科院分区：
文献类型：
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作者：
R. Smith;C. Baglioni
采用Sephadex G-75凝胶过滤层析法分离天然人和重组人、鼠肿瘤坏死因子（TNF）。TNF的活性形式是通过其与HeLa细胞的受体结合试验中的抑制活性来鉴定的，并被洗脱为Mr约为55,000的蛋白质。通过凝胶过滤将放射性的人和鼠TNF分离成Mr约为55,000的主峰，对应于三聚体，Mr约为17,000的小峰，对应于单体。结合实验表明，该计时器的活性至少是单体的8倍。在加入非离子清洁剂Triton X-100后，人TNF部分解离成单体。分离的单体具有较低的结合亲和力（KD = 70 nM）和较低的细胞毒性，而三聚体具有较高的结合亲和力（KD = 90 pM）和细胞毒性。当125I-TNF与细胞结合时，没有检测到单体的释放，这表明该三聚体可以直接与细胞受体结合而不解离成亚基。通过将125I-TNF三聚体与双[2-（琥珀酰亚胺ooxycarbonyloxy）乙基]砜交联，获得了这种结合的进一步证据。这些三聚体与HeLa细胞结合，可以与细胞受体分离，并引起细胞毒性反应。这些结果表明，三聚体，无论是天然的还是交联的，都与受体结合，是TNF的生物活性形式。
Natural human and recombinant human and murine tumor necrosis factors (TNF) were fractionated by gel filtration chromatography on Sephadex G-75. The active form of TNF was identified by its inhibitory activity in receptor binding assays with HeLa cells and was eluted as a protein of Mr approximately 55,000. Radioiodinated human and murine TNF were fractionated by gel filtration into a major peak of Mr approximately 55,000, corresponding to a trimer, and a minor peak of Mr approximately 17,000, corresponding to a monomer. Binding assays showed that the timer was at least 8-fold more active than the monomer. The human TNF partially dissociated into monomers upon addition of the nonionic detergent Triton X-100. Isolated monomers showed low binding affinity (KD = 70 nM) and reduced cytotoxicity, whereas trimers showed high binding affinity (KD = 90 pM) and cytotoxicity. When 125I-TNF was bound to cells, no release of monomer was detectable, suggesting that the trimer could directly bind to cellular receptors without dissociating into subunits. Further evidence for such binding was obtained by cross-linking 125I-TNF trimers with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. These trimers were bound to HeLa cells, could be dissociated from cellular receptors, and elicited a cytotoxic response. These results show that trimers, whether native or cross-linked, bind to receptors and are the biologically active form of TNF.