Galizzi J P, Castle B, Djossou O, Harada N, Cabrillat H, Yahia S A, Barrett R, Howard M, Banchereau J
Laboratory for Immunological Research, Dardilly, France.
J Biol Chem. 1990 Jan 5;265(1):439-44.
The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.
白细胞介素4（IL-4）受体是从长臂猿T细胞系MLA 144中纯化得到的。发现这些细胞表达大量的人IL-4结合蛋白（5000 - 6000个位点/细胞），其亲和常数（Kd）与人细胞系中测得的相似（Kd = 40 - 70 pM）。125I-IL-4与人细胞系和MLA 144细胞的亲和交联显示标记了三种分子量约为130、75和65 kDa的蛋白质。使用Triton X-100从MLA 144细胞中溶解人IL-4结合位点，然后通过羧甲基色谱法进行纯化，该方法去除了50%的蛋白质而不损失IL-4结合活性。然后依次在麦胚凝集素和单个IL-4 Affi-Gel 10柱上进行亲和纯化，最终使IL-4受体纯化了8000倍。当在银染的十二烷基硫酸钠-聚丙烯酰胺凝胶上进行分析时，纯化的受体迁移为一条单一的分子量为130±5 kDa的分子条带。通过交联实验以及在将纯化的受体在十二烷基硫酸钠-聚丙烯酰胺凝胶上进行电泳转移后125I-IL-4与硝酸纤维素膜的特异性结合，证明了130-kDa的蛋白质就是IL-4受体。
