Sluss P M, Reichert L E
Biol Reprod. 1984 Jun;30(5):1091-104. doi: 10.1095/biolreprod30.5.1091.
Porcine follicular fluid (PFF) inhibited the binding of 125I-human follicle-stimulating hormone (hFSH) to receptor in vitro in a dose-dependent fashion. PFF (2.5 l) was fractionated on the basis of apparent molecular weight (Mr) by ultrafiltration using hollow fibers and membranes of precalibrated pore size. Desalted, low Mr (500-5000) subfractions containing FSH-binding inhibitor (FSH-BI) activity were further purified by Sephadex G10 gel filtration and anion-exchange high-performance liquid chromatography (HPLC). This resulted in the partial purification of several low Mr FSH-BIs. Three major peaks of FSH-BI were resolved on the Sephadex G10 column eluted with water; G10-1 [elution volume (Ve)/exclusion volume (Vo) = 1.1] had only FSH-BI activity, while G10-2 (Ve/Vo = 1.4) and G10-3 (Ve/Vo = 1.5) had both FSH-BI and luteinizing hormone (LH)-BI activities. A fourth strongly retarded peak (G10-4; Ve/Vo = 2.7) was also obtained. This latter fraction had only FSH-BI activity and represented less than 1% of the FSH-BI activity applied to the column. No separation of these fractions was obtained when the column was eluted with 10 mM ammonium acetate instead of water, suggesting resolution was due to ion-exchange or hydrophobic interactions with the Sephadex. Anion-exchange (Polyanion SI) HPLC of G10-1, G10-2 or G10-3 samples resolved several fractions with FSH-BI activity. A fraction unretained at either pH 5.0 or 7.0 (HPLC-1) was present in all samples. A fraction strongly retained by the column (HPLC-2) and a fraction eluted between 0.13 to 0.24 M acetate (HPLC-3) were present in G10-1 and G10-2 but not in G10-3. HPLC-4, eluted between 0.32 to 0.36 M acetate at pH 5.0, was detected only in G10-3 samples. The most potent low Mr FSH-BI obtained (HPLC-2) inhibited FSH binding by 50% at a dose of 10 micrograms and was enriched approximately 2500-fold relative to whole follicular fluid. These results indicate that PFF contains several low (500-5000) Mr inhibitors of FSH binding to receptor in vitro which differ on the basis of charge, hormone specificity and possibly molecular size and hydrophobicity.
猪卵泡液（PFF）在体外以剂量依赖的方式抑制125I-人促卵泡激素（hFSH）与受体的结合。使用中空纤维和预先校准孔径的膜通过超滤对2.5升PFF进行基于表观分子量（Mr）的分级分离。含有促卵泡激素结合抑制剂（FSH-BI）活性的脱盐低Mr（500-5000）亚组分通过Sephadex G10凝胶过滤和阴离子交换高效液相色谱（HPLC）进一步纯化。这导致了几种低Mr FSH-BI的部分纯化。在用水洗脱的Sephadex G10柱上解析出三个主要的FSH-BI峰；G10-1[洗脱体积（Ve）/排阻体积（Vo）=1.1]仅具有FSH-BI活性，而G10-2（Ve/Vo = 1.4）和G10-3（Ve/Vo = 1.5）同时具有FSH-BI和促黄体生成素（LH）-BI活性。还获得了第四个强烈滞留峰（G10-4；Ve/Vo = 2.7）。后一个级分仅具有FSH-BI活性，占应用于柱的FSH-BI活性的不到1%。当用10 mM醋酸铵而不是水洗脱柱时，未获得这些级分的分离，表明分离是由于与Sephadex的离子交换或疏水相互作用。对G10-1、G10-2或G10-3样品进行阴离子交换（聚阴离子SI）HPLC解析出几个具有FSH-BI活性的级分。所有样品中都存在在pH 5.0或7.0时均未保留的一个级分（HPLC-1）。G10-1和G10-2中存在被柱强烈保留的一个级分（HPLC-2）和在0.13至0.24 M醋酸盐之间洗脱的一个级分（HPLC-3），而G10-3中不存在。仅在G10-3样品中检测到在pH 5.0时于0.32至0.36 M醋酸盐之间洗脱的HPLC-4。获得的最有效的低Mr FSH-BI（HPLC-2）在剂量为10微克时抑制FSH结合50%，相对于全卵泡液富集了约2500倍。这些结果表明，PFF含有几种低（500-5000）Mr的体外抑制FSH与受体结合的抑制剂，它们在电荷、激素特异性以及可能的分子大小和疏水性方面存在差异。
