Isolation of various isoforms in inhibin-related proteins and their biological activity.

抑制素是一种糖蛋白，由两个不同的亚基组成，称为α和β，它们通过二硫键连接在一起。激活素是一种β亚基二聚体，由抑制素βA和BETAB亚基组成，分别称为激活素A、激活素AB和激活素B。在小实验动物中，如对抑制素的生物学和免疫学实验最有用的大鼠，是不可能分离和鉴定抑制素的。因此，我们改进了抑制素、激活素和卵泡抑素的纯化方法。免疫亲和层析法是从卵泡液中分离纯化抑制素最常用的方法，由于在人卵泡液中未检测到抑制素B，因此我们尝试从人睾丸cDNA文库中克隆出编码人抑制素α和BETAB亚基的重组人抑制素B。将cDNA连接到表达载体上，并将其导入中国仓鼠卵巢细胞。将α亚基和BETAB亚基的cDNAs导入细胞，可分泌抑制素B和激活素B。采用多克隆抗体免疫亲和层析的方法，对培养基中低水平的抑制素B进行纯化。激活素/FSP复合体经硫酸纤维素亲和层析、肝素琼脂糖凝胶层析和Superdex PG S20凝胶渗透层析纯化。激活素/FSP复合体进一步经反相高效液相色谱分离，得到六个主要组分。前三个组分具有抑制FSH的活性。其他组分对培养的垂体细胞具有促卵泡激素刺激活性，分别对应于激活素-A、-AB和B。

Inhibin is a glycoprotein consisting of two dissimilar subunits, termed alpha and beta, that are linked by disulfide bounds. Activin is a beta-subunit dimer and consists of inhibin betaA and betaB subunits that have been termed activin A,activin AB,and activin B.It is impossible to isolate and characterize the inhibin in small experimental animals such as the rat that is the most useful for biological and immunological experiments on inhibin. Therefore, we improved the methods for purifying inhibin, activin and follistatin. Immunoaffinity chromatography is the most useful applied method for the purification of inhibin from follicular fluid.Since inhibin B was not detected in human follicular fluid, we tried to produce recombinant human inhibin B.cDNA clones encoding human inhibin alpha- and betaB subunits were isolated from a human testicular cDNA library. The cDNAs were linked to an expression vector and transfected into Chinese hamster ovarian cells. The cells transfected with the alpha-subunit and betaB-subunit cDNAs secreted inhibin B and activin B.Immunoaffinity chromatography with polyclonal antibodies was used to purify the low levels of inhibin B contained in the culture medium as described above.In the process of purifying bovine inhibin by immunoaffinity chromatography, the flow through fraction from the immunoaffinity column was collected. The activin/FSP complex was then purified through successive steps of affinity chromatography with sulfated cellulose and heparin Sepharose, and FPLC gel permeation on a Superdex pg S20 column. Activin/FSP complex, was further subjected to reverse-phase HPLC providing six major fractions. The first three fractions had FSH inhibiting activity. The other fractions had FSH stimulating activity with cultured pituitary cells that correspond to activin-A,-AB,and B,respectively.