Shin J, Ji T H
J Biol Chem. 1985 Nov 15;260(26):14020-5.
A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.
人促卵泡激素的一种光可激活衍生物被用于鉴定猪颗粒细胞上的促卵泡激素受体。该激素与一种异双功能试剂4-叠氮苯甲酰甘氨酸的N-羟基琥珀酰亚胺酯缩合，并进行放射性碘化。125I标记的激素（125I-激素）衍生物与受体结合的数量与125I-激素本身相同，但解离常数略低，125I-激素衍生物的解离常数为1.12×10(10) M-1，而125I-激素的解离常数为1.4×10(10) M-1。未处理的激素可阻断这种结合。其α和β亚基可通过光解交联产生αβ二聚体。当结合到细胞上的125I-激素衍生物进行光解交联，产物在还原条件下于十二烷基硫酸钠-聚丙烯酰胺凝胶上进行电泳分离时，除了α、β和αβ条带外，还出现了两条电泳迁移率较低的新条带（106和61 kDa）。这些交联复合物的形成需要光解，且125I-激素衍生物特异性结合到带有受体的细胞上。过量未处理的促卵泡激素可阻断结合，但人绒毛膜促性腺激素和促甲状腺激素则不能。在非还原条件下，出现一条主要的交联复合物条带（104 kDa）。用二硫苏糖醇还原并进行二维电泳后，104-kDa条带产生了75和61 kDa的两个较小复合物，表明损失了两个成分且存在成分间二硫键。成功产生104-kDa复合物需要用N-乙基马来酰亚胺阻断游离巯基。然而，它与各种蛋白酶抑制剂、激素与细胞或质膜部分孵育的温度和时间段无关。讨论了光亲和标记成分的质量估计及其与激素的相互作用。
