Eid P, Bandu M T, Uze G, Mogensen K E
Laboratoire d'Oncologie Virale, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
Eur J Biochem. 1988 Feb 1;171(3):675-82. doi: 10.1111/j.1432-1033.1988.tb13839.x.
The Burkitt-derived line, Daudi, whose proliferation is inhibited by human alpha-interferon (IFN-alpha), was treated with 125I-labelled recombinant human IFN-alpha A. After separation from unbound ligand, cell-bound IFN was extracted with the detergent digitonin yielding soluble and insoluble complexes of IFN and receptor, together with a certain amount of uncomplexed IFN. 1. Soluble complexes were stable enough to be separated from uncomplexed IFN by permeation chromatography. Treatment of soluble complexes with the bifunctional reagent, disuccinimidyl suberate, yielded a radioactive product separating with an Mr of 130,000 on electrophoresis in sodium dodecyl sulphate. Similar complexes could be recovered with sodium dodecyl sulphate from the digitonin-insoluble residue, treated with the bi-functional reagent. 2. The total (soluble and insoluble) of complexed IFN obtained after digitonin extraction was a constant fraction (0.62) of the total cell-bound radioactivity, being independent of the concentration of IFN added to the cells (less than pM to greater than nM), and of the time of incubation (1 min to 20 h). However, between 30 min and 3 h of incubation, the insoluble complex increased, at the expense of the soluble complex, and there appeared a cellular pool of degraded ligand. From 3 h to 20 h the distribution of ligand-derived radioactivity remained constant while the total amount decreased to less than 10% of its value at 30 min. This decrease in binding was matched by the appearance of an equivalent quantity of radiolabelled fragments in the culture medium. 3. The inhibition of cellular division due to IFN was shown to be coincident with the disappearance of cellular binding and with the cell-mediated degradation of receptor-complexed IFN. We propose that IFN removes its own receptor and, in doing so, blocks a linked function necessary for the stimulated growth of Daudi cells.
源自伯基特淋巴瘤的细胞系Daudi，其增殖受到人α干扰素（IFN-α）的抑制，用125I标记的重组人IFN-α A处理该细胞系。从未结合的配体中分离出来后，用去污剂洋地黄皂苷提取细胞结合的IFN，得到IFN与受体的可溶性和不溶性复合物，以及一定量未复合的IFN。1. 可溶性复合物足够稳定，可通过渗透色谱从未复合的IFN中分离出来。用双功能试剂辛二酸二琥珀酰亚胺酯处理可溶性复合物，在十二烷基硫酸钠中电泳时产生一种放射性产物，其相对分子质量为130,000。用双功能试剂处理洋地黄皂苷不溶性残渣后，也可用十二烷基硫酸钠回收类似的复合物。2. 洋地黄皂苷提取后获得的复合IFN的总量（可溶性和不溶性）是细胞结合的总放射性的一个恒定分数（0.62），与添加到细胞中的IFN浓度（小于pM至大于nM）以及孵育时间（1分钟至20小时）无关。然而，在孵育30分钟至3小时之间，不溶性复合物增加，可溶性复合物减少，并且出现了一个细胞内降解配体的池。从3小时至20小时，配体衍生的放射性分布保持恒定，而总量降至30分钟时其值的不到10%。这种结合的减少与培养基中出现等量的放射性标记片段相匹配。3. 已证明IFN对细胞分裂的抑制与细胞结合的消失以及细胞介导的受体复合IFN的降解同时发生。我们提出，IFN去除其自身的受体，在此过程中，阻断了Daudi细胞刺激生长所必需的一个相关功能。
