Fukunaga R, Ishizaka-Ikeda E, Nagata S
Osaka Bioscience Institute, Japan.
J Biol Chem. 1990 Aug 15;265(23):14008-15.
A receptor for mouse granulocyte colony-stimulating factor (G-CSF) has been found on the cell surface of mouse myeloid leukemia cell line NFS-60. Chemical cross-linking of the receptor with radioiodinated G-CSF, followed by gel electrophoresis in the presence of sodium dodecyl sulfate, has revealed that the G-CSF receptor in the NFS-60 cells is a single polypeptide of Mr approximately 100,000-130,000. The receptor in the membrane fraction of NFS-60 cells were solubilized in an active form with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid. The solubilized receptor was purified approximately 100,000-fold to near homogeneity using a G-CSF affinity gel and gel filtration on a Superose 12 column, as measured by the selective precipitation of the 125I-G-CSF-receptor complex by polyethylene glycol. The purified G-CSF receptor has two classes of binding characteristics, one with an equilibrium dissociation constant (Kd) of 120-360 pM which is comparable with the Kd value for the cell-surface receptor, and the other with a higher Kd value of 2.6-4.2 nM. Analyses of the purified receptor by ligand blotting and sucrose density gradient centrifugation indicated that the low-affinity receptor is the monomer of the Mr 100,000-130,000 protein, whereas the high-affinity receptor consists of oligomers of the protein.
在小鼠髓系白血病细胞系NFS - 60的细胞表面发现了小鼠粒细胞集落刺激因子（G - CSF）的受体。用放射性碘化的G - CSF对该受体进行化学交联，随后在十二烷基硫酸钠存在下进行凝胶电泳，结果显示NFS - 60细胞中的G - CSF受体是一种分子量约为100,000 - 130,000的单一多肽。NFS - 60细胞膜部分的受体用3 - [(3 - 胆酰胺丙基)二甲基铵基]-1 - 丙烷磺酸盐以活性形式溶解。通过聚乙二醇对125I - G - CSF - 受体复合物进行选择性沉淀测定，用G - CSF亲和凝胶和Superose 12柱上的凝胶过滤将溶解的受体纯化了约100,000倍，达到接近均一的程度。纯化的G - CSF受体具有两类结合特性，一类的平衡解离常数（Kd）为120 - 360 pM，与细胞表面受体的Kd值相当，另一类的Kd值较高，为2.6 - 4.2 nM。通过配体印迹和蔗糖密度梯度离心对纯化受体进行分析表明，低亲和力受体是分子量为100,000 - 130,000蛋白质的单体，而高亲和力受体由该蛋白质的寡聚体组成。
