Das M, Miyakawa T, Fox C F, Pruss R M, Aharonov A, Herschman H R
Proc Natl Acad Sci U S A. 1977 Jul;74(7):2790-4. doi: 10.1073/pnas.74.7.2790.
A photoreactive derivative of epidermal growth factor (EGF) has been used to identify and specifically label a membrane receptor for EGF on mouse 3T3 cells. Photoactivable EGF, labeled with 125I, was incubated with 3T3 cells and then photolyzed in situ to generate a nitrene capable of reacting with a wide variety of chemical bonds. Analysis of the system by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed, besides the band of EGF, only one other major radioactive band, at a position indicating an apparent molecular weight of 190,000. This band was absent when a nonresponsive and nonbinding variant of 3T3 was used. A direct proportionality between binding activity and crosslinked complex formation was demonstrated using a variety of binding conditions. "Down regulated" cells, in which EGF binding activity was greatly reduced by prolonged incubation with an appropriate concentration of EGF, also had a decrease in covalent complex formation proportional to the decrease in EGF binding activity.
表皮生长因子（EGF）的一种光反应性衍生物已被用于鉴定并特异性标记小鼠3T3细胞上的EGF膜受体。用125I标记的可光活化EGF与3T3细胞一起孵育，然后原位光解以产生能够与多种化学键反应的氮烯。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳对该系统进行分析，结果显示，除了EGF条带外，仅在一个表观分子量为190,000的位置出现另一条主要放射性条带。当使用无反应且不结合的3T3变体时，该条带不存在。使用多种结合条件证明了结合活性与交联复合物形成之间存在直接比例关系。“下调”细胞，即通过用适当浓度的EGF长时间孵育使EGF结合活性大大降低的细胞，其共价复合物形成也随着EGF结合活性的降低而成比例减少。
