Shin J, Ji T H
J Biol Chem. 1985 Oct 15;260(23):12828-31.
The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.
放射性碘化促卵泡激素（FSH）的α和β亚基以及αβ二聚体的电泳迁移率会根据二硫苏糖醇等还原剂的浓度而发生显著变化。β亚基的变化比α亚基更为显著。与猪颗粒细胞上或Triton X-100提取物中的受体结合的125I-FSH，用可裂解（非二硫键）的同型双功能试剂进行交联，在不使用还原剂的情况下用十二烷基硫酸钠溶解，然后进行电泳。交联后的样品除了激素亚基和二聚体条带外，还显示出三条高分子质量条带。质量最轻的条带为110 kDa，是主要条带，另外两条76 kDa和62 kDa的条带几乎难以察觉。用二硫苏糖醇还原后，110 kDa条带减少，而76 kDa和62 kDa条带增加，表明110 kDa复合物各组分之间存在二硫键。二硫键连接复合物的形成需要特异性结合激素受体并进行交联的125I-FSH，过量的天然FSH可阻止其形成，但人绒毛膜促性腺激素则不能。复合物的形成与用N-乙基马来酰亚胺封闭游离巯基无关。当交联复合物在凝胶基质中还原并在新鲜凝胶上分析时，76 kDa和62 kDa的复合物由110 kDa条带产生，表明损失了两个组分。估计损失的组分分别为14 kDa和34 kDa。交联复合物的形成和裂解速率表明，22 kDa、14 kDa和34 kDa的组分依次逐步添加到FSHαβ二聚体上。交联复合物还原的结果证明了这三个组分之间存在二硫键连接。
