Shin J, Ji T H
J Biol Chem. 1985 Oct 15;260(23):12822-7.
Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with 125I-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.
完整的人促卵泡激素（FSH）的α和β亚基均用125I-碘化钠和氯胺-T进行放射性碘化，并可在十二烷基硫酸钠-聚丙烯酰胺凝胶上分离。放射性碘化的FSH与一种可裂解的（非二硫键）同型双功能试剂在猪颗粒细胞表面与它的膜受体以及与受体的Triton X-100溶解形式进行亲和交联。交联样品除了激素条带外，还显示出另外三条电泳迁移率较慢的条带，分别对应于65、83和117 kDa。激素αβ二聚体条带对应于43 kDa。这三条带的形成需要125I-激素与受体特异性结合并随后进行交联。过量的天然激素可阻止结合，但其他激素则不能。交联试剂的单功能类似物未能产生这三条带。试剂浓度依赖性交联表明它们的形成是顺序性的；较小的复合物先形成，然后是较大的复合物。当交联复合物的凝胶经处理以裂解共价交联，然后在第二维进行电泳时，除了激素的α和β亚基外，还释放出18、22和34 kDa的成分。
