Isolation of Inhibin Receptor and Siginal Transuduction In TGF-β Superfamily

抑制素是一种二硫化物连接的二聚体糖蛋白，由α亚基和β亚基组成。激活素是由抑制素β亚基组成的二硫键二聚体蛋白。虽然激活素结合蛋白卵泡抑素可以修饰激活素的活性，但对抑制素的生理结合蛋白知之甚少。采用生物素化抑制素作为抑制素-抑制素结合蛋白复合物的配体印迹探针，采用抑制素α亚基免疫亲和层析、sepphacryl S 200 pg凝胶渗透层析和反相高效液相色谱法对牛卵泡液中43 kDa抑制素结合蛋白进行了纯化。该蛋白的nh2末端氨基酸序列与人抑制素/激活素βA亚基的部分前体序列相同。以eu标记的抑制素和激活素为配体，采用固相结合法研究纯化蛋白的结合能力。Eu标记抑制素与43 kDa蛋白的结合亲和力是所研究的卵泡抑制素的4倍左右；猪卵泡素288，猪卵泡素303，猪卵泡素315，或重组人卵泡素288。43 kDa蛋白与激活素的最大结合量与follistatin相同，43 kDa蛋白与激活素的亲和力约为288 follistatin的一半。研究了43 kDa蛋白对垂体细胞培养FSH释放和K562细胞血红蛋白生物合成的影响。43 kDa蛋白对大鼠垂体前叶细胞FSH释放的抑制曲线与pFS288基本一致。激活素对促卵泡刺激素释放有刺激作用，43 kDa蛋白对促卵泡刺激素释放有剂量依赖性抑制。在K562细胞血红蛋白生物合成的研究中，激活素刺激的血红蛋白生物合成和43 kDa蛋白对激活素刺激的血红蛋白生物合成表现出剂量依赖性抑制。

Inhibin is a disulfide-linked, dimeric glycoprotein composed of an α subunit and β subunits. Activin is disulfide-linked dimeric protein comprised of inhibin β subunits. Although the activity of activin is modified by activin binding protein, follistatin, little is known about the physiological binding proteins of inhibin.Using biotinylated inhibin as the probe of ligand blotting of inhibin-inhibin binding protein complexes, 43 kDa inhibin binding protein was identified in bovine follicular fluid by sequential chromatographic purification by immunoaffinity chromatography against inhibin α subunit, Sephacryl S 200 pg gel permeation chromatography, and RP-HPLC.NH2-terminal amino acid sequencing of the protein was identical to a part of the precursor sequence of human inhibin/activin βA subunit. The binding ability of the purified protein was studied with solid phase binding assay using Eu-labeled inhibin and activin as ligands. The binding affinity of Eu labeled inhibin to 43 kDa protein was about four times stronger than that to any follistatins studied ; porcine follistatin 288, porcine follistatin 303, porcine follistatin 315, or recombinant human follistatin 288. The maximal-binding of 43 kDa protein to activin was identical to follistatin and the affinity of 43 kDa protein to activin was about a half of 288 follistatin. The effect of 43 kDa protein on FSH release in pituitary cell culture and hemoglobin biosynthesis in K562 cells was studied. 43 kDa protein showed almost the same suppression curve with pFS288 on the FSH release in rat anterior pituitary cell culture. FSH release was stimulated by the addition of activin in the culture, and the stimulated release of FSH was suppressed by 43 kDa protein dose dependently. In the study of hemoglobin biosynthesis of K562 cells, activin stimulated hemoglobin biosynthesis and 43 kDa protein showed dose dependent suppression of hemoglobin biosynthesis stimulated by activins.