TUMOR NECROSIS FACTOR - SPECIFIC BINDING AND INTERNALIZATION IN SENSITIVE AND RESISTANT CELLS
TUMOR NECROSIS FACTOR - SPECIFIC BINDING AND INTERNALIZATION IN SENSITIVE AND RESISTANT CELLS
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DOI：
10.1073/pnas.82.22.7626
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
VILCEK, J
中科院分区：
文献类型：
--
作者：
TSUJIMOTO, M;YIP, YK;VILCEK, J
高度纯化的，大肠杆菌衍生的重组人肿瘤坏死因子（TNF）标记125碘，并用于确定受体结合，内化，并在小鼠L929细胞（高度敏感的TNF细胞毒性作用）和二倍体人FS-4细胞（抗TNF细胞毒性）的细胞内降解。125 I标记的TNF与L929和FS-4细胞上的高亲和力受体特异性结合。结合数据的Scatchard分析表明每个L929细胞存在2200个结合位点，每个FS-4细胞存在7500个结合位点。计算出的离解常数为6.1 × 10 - 4。10-10 M和3.2 ×对于L929和FS-4细胞分别为10-10 M。在L929和FS-4细胞中，在37 ℃孵育。C导致大量的细胞结合的TNF的快速内化，随后在组织培养基中出现三氯乙酸可溶性125 I放射性，由于TNF的降解。在L929和FS-4细胞中，氯喹（一种溶酶体蛋白酶抑制剂）的存在下，TNF的降解而非细胞摄取受到抑制，这表明降解发生在细胞内，可能在溶酶体内。这些结果表明，FS-4细胞对TNF细胞毒性的抗性不是由于缺乏受体或它们不能内化和降解TNF。
Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 125I and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 125I-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 .times. 10-10 M and 3.2 .times. 10-10 M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 37.degree. C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF.