Furukawa K, Yamoto M, Kokawa N, Nakano R
Department of Obstetrics and Gynecology, Wakayama Medical College, Japan.
Eur J Endocrinol. 1994 Jun;130(6):625-33. doi: 10.1530/eje.0.1300625.
We performed the purification of high-molecular-weight follicle-stimulating hormone binding inhibitor (FSHBI) from porcine follicular fluids. The FSHBI activities of high-molecular-weight fractions acquired by ultrafiltration of follicular fluids from small, medium and large follicles with Centriflo CF25 membrane cone were 277.2 +/- 24.6, 176.7 +/- 3.0 and 141.3 +/- 3.6 U, respectively. By affinity chromatography of CF25 retentate with a column of Blue Sepharose CL6B, 94.1 +/- 5.3% of FSHBI activity was recovered in the unretained fraction. The FSHBI in the unretained fraction was purified by anion-exchange chromatography with a column of Mono Q and gel filtration on Sephacryl S300HR. As a result of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the final purified fraction, a single silver-stained band was observed at 310 kD under the non-reducing conditions. On the other hand, under the reducing conditions, SDS-PAGE revealed three bands at 178, 101 and 55 kD. A double reciprocal plot analysis of this substance showed competitive inhibition in FSH binding. The results of the present study suggest the existence of a 310 kD FSHBI composed of three subunits of 178, 101 and 55 kD in porcine follicular fluids.
我们从猪卵泡液中纯化了高分子量促卵泡激素结合抑制剂（FSHBI）。使用Centriflo CF25膜锥对来自小、中、大卵泡的卵泡液进行超滤，所获得的高分子量级分的FSHBI活性分别为277.2±24.6、176.7±3.0和141.3±3.6 U。用Blue Sepharose CL6B柱对CF25截留物进行亲和层析，94.1±5.3%的FSHBI活性在未保留级分中回收。未保留级分中的FSHBI通过用Mono Q柱进行阴离子交换层析和在Sephacryl S300HR上进行凝胶过滤来纯化。对最终纯化级分进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）的结果显示，在非还原条件下，在310 kD处观察到一条单一的银染带。另一方面，在还原条件下，SDS-PAGE显示在178、101和55 kD处有三条带。对该物质进行双倒数作图分析表明其对FSH结合具有竞争性抑制作用。本研究结果表明，猪卵泡液中存在一种由178、101和55 kD的三个亚基组成的310 kD FSHBI。
