Chiba S, Shibuya K, Miyazono K, Tojo A, Oka Y, Miyagawa K, Takaku F
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
J Biol Chem. 1990 Nov 15;265(32):19777-81.
The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-125I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.
人粒细胞巨噬细胞集落刺激因子（GM-CSF）受体α链是该受体的低亲和力组分，利用生物素化的GM-CSF将其从人胎盘中溶解并亲和纯化。对125I-GM-CSF与胎盘膜提取物的结合进行Scatchard分析表明，GM-CSF受体的解离常数（Kd）为0.5 - 0.8 nM，这与完整胎盘膜上GM-CSF受体α链的Kd值相对应。使用光反应性交联剂N-羟基琥珀酰亚胺-4-叠氮基苯甲酸酯（HSAB）对溶解的蛋白进行亲和标记，显示出一条70 - 95 kDa的单一特异性条带，代表配体-受体复合物。约2 g胎盘膜提取物通过生物素化GM-CSF固定的链霉亲和素-琼脂糖柱，在十二烷基硫酸钠银染凝胶上产生一条70 kDa的主要条带。对纯化材料进行放射性碘化显示，纯化蛋白的近似分子量为70 kDa，等电点为6.6。使用HSAB-125I-GM-CSF通过光亲和标记证明了纯化材料的结合活性，产生了一条与粗蛋白类似的70 - 95 kDa特异性条带。
