Purification and characterization of a human tumor necrosis factor from the LuKII cell line.
Purification and characterization of a human tumor necrosis factor from the LuKII cell line.
复制标题
从 LuKII 细胞系中纯化和表征人肿瘤坏死因子。
DOI：
10.1073/pnas.82.19.6637
复制
发表时间：
1985
影响因子：
11.1
通讯作者：
Old,LJ
中科院分区：
文献类型：
--
作者：
Rubin,BY;Anderson,SL;Sullivan,SA;Williamson,BD;Carswell,EA;Old,LJ
从人淋巴母细胞系LuKII产生的具有肿瘤坏死因子活性的因子[命名为TNF(LuKII)]，经控制孔玻璃层析、扁豆凝集素-琼脂糖柱层析和蛋白红色琼脂糖层析依次纯化，得到比活性为1.5×10(7)单位/毫克蛋白质的肿瘤坏死因子，等电点约为6.7。用NaDodSO4/PAGE分离纯化的肿瘤坏死因子(LuKII)在还原和非还原条件下均含有7条蛋白带，分别为80,000，70,000，43,000，25,000，23,000，21,000和19,000。MR 80,000和70,000的蛋白质不能解离为低分子量组分。多肽图谱分析和免疫印迹分析表明，纯化的肿瘤坏死因子(LuKII)制剂中的7条蛋白条带是相关的。在还原条件下用NaDodSO4/PAGE对肿瘤坏死因子(LuKII)进行分级后，在Mr 70,000和19,000-25,000区域恢复了肿瘤坏死因子的活性。纯化的人肿瘤坏死因子(LuKII)(I)在标准的体内小鼠肿瘤坏死试验中可产生Meth A小鼠肉瘤的出血性坏死；(Ii)对一组人癌细胞具有与小鼠肿瘤坏死因子相同的反应模式(细胞毒性/细胞抑制/无作用)；以及(Iii)其抗细胞作用被干扰素增强，这也是小鼠肿瘤坏死因子的一个特征。
A factor with tumor necrosis factor (TNF) activity produced by the LuKII human lymphoblastoid cell line [designated TNF(LuKII)] was purified sequentially by using controlled-pore glass, lentil lectin-Sepharose, and procion red agarose chromatography, yielding TNF with a specific activity of 1.5 X 10(7) units per mg of protein and an isoelectric point of approximately equal to 6.7. Purified TNF(LuKII) fractionated by NaDodSO4/PAGE under reducing as well as nonreducing conditions was found to contain seven protein bands of Mr 80,000, 70,000, 43,000, 25,000, 23,000, 21,000, and 19,000. The proteins of Mr 80,000 and 70,000 could not be dissociated into lower molecular weight components. Peptide mapping analysis and immunoblotting analysis revealed that the seven protein bands in the purified TNF(LuKII) preparations are related. After fractionation of TNF(LuKII) by NaDodSO4/PAGE under reducing conditions, TNF activity was recovered from the regions of Mr 70,000 and 19,000-25,000. Purified human TNF(LuKII) (i) produces hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay; (ii) has the same pattern of reactivity as mouse TNF (cytotoxic/cytostatic/no effect) on a panel of human cancer cell lines; and (iii) has its anticellular effect potentiated by interferon, also a feature of mouse TNF.