Evidence that the bovine ovary secretes large amounts of monomeric inhibin alpha subunit and its isolation from bovine follicular fluid.
Evidence that the bovine ovary secretes large amounts of monomeric inhibin alpha subunit and its isolation from bovine follicular fluid.
复制标题
牛卵巢分泌大量单体抑制素α亚基的证据及其从牛卵泡液中的分离。
DOI：
10.1677/jme.0.0020189
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发表时间：
1989
影响因子：
3.5
通讯作者：
Raquel Castillo
中科院分区：
文献类型：
--
作者：
P. G. Knight;A. Beard;J. Wrathall;Raquel Castillo
使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 结合灵敏的免疫印迹程序对牛卵泡液 (FF) 进行分析，解析了与针对人抑制素 α 亚基 (hI α(1-32]) n 末端的抗血清发生免疫反应的几种成分。在非还原条件下，三个强烈染色的条带的表观 Mr 值为 116,000，存在 44,000 和 25,000，而在还原条件下，仅检测到两条强烈染色的条带（Mr 43,000 和 21,000）。使用琼脂糖珠对样品（40 ml）进行免疫亲和浓缩后，在牛子宫卵巢静脉和外周静脉血浆中也发现了 Mr 44,000 和 25,000 免疫反应形式（非还原条件）。与抗 hI α(1-32) 偶联、SDS-PAGE 和免疫印迹法通过凝胶渗透色谱 (Sephacryl S-200)、免疫亲和色谱 (Sepharose-anti-hI α(1-32)) 和反相高效液相色谱 (RP-HPLC；使用 C18 和 C8 柱从 bFF（30 ml，19.5 g 蛋白质）中分离出 750 微克蛋白质，通过 RP-HPLC 和 SDS-PAGE 检测，该蛋白质基本均质，Mr 为 25,000（非还原条件）/21,000（还原条件），与牛 FF、子宫卵巢静脉血浆、外周血浆和 GCCM 中发现的最低 Mr 的免疫反应成分相同。分离的材料与针对 hI α(1-32) 和纯化的 Mr 32,000 牛抑制素的抗血清具有高度免疫反应性，但在大鼠垂体细胞抑制素生物测定中进行测试时，其缺乏生物活性，显示其氨基酸序列（残基 1-14）与其他地方报道的牛抑制素 α 亚基（Mr 20,000/21,000）相同。总之，本研究表明，牛卵巢分泌大量的单体抑制素α亚基，这种物质在外周血中的意外存在可能会阻碍使用传统放射免疫测定法获取牛体内抑制素循环水平的生理相关数据。
Analysis of bovine follicular fluid (FF) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with a sensitive immunoblotting procedure resolved several components that were immunoreactive with an antiserum directed against the n-terminus of the alpha subunit of human inhibin (hI alpha(1-32]. Under non-reducing conditions, three intensely stained bands having apparent Mr values of 116,000, 44,000 and 25,000 were present, whilst under reducing conditions only two intensely stained bands (Mr 43,000 and 21,000) were detected. The Mr 44,000 and 25,000 immunoreactive forms (non-reducing conditions) were also demonstrated in bovine utero-ovarian vein and peripheral venous plasma after subjecting samples (40 ml) to immunoaffinity concentration using Sepharose beads coupled to anti-hI alpha(1-32), SDS-PAGE and immunoblotting. The same approach revealed the presence of the smaller (Mr 25,000) form in bovine granulosa cell-conditioned culture medium (GCCM). Gel-permeation chromatography (Sephacryl S-200), immunoaffinity chromatography (Sepharose-anti-hI alpha(1-32] and reversed-phase high-performance liquid chromatography (RP-HPLC; C18 and C8 columns) were employed to isolate from bFF (30 ml, 19.5 g protein) 750 micrograms protein which appeared essentially homogeneous by RP-HPLC and SDS-PAGE and had an Mr of 25,000 (non-reducing conditions)/21,000 (reducing conditions), identical to that of the immunoreactive component of lowest Mr found in bovine FF, utero-ovarian vein plasma, peripheral plasma and GCCM. The isolated material was highly immunoreactive with antisera against both hI alpha(1-32) and purified Mr 32,000 bovine inhibin but was devoid of biological activity when tested in a rat pituitary cell inhibin bioassay. Amino-terminal analysis revealed an amino acid sequence (residues 1-14) identical to that reported elsewhere for the alpha subunit (Mr 20,000/21,000) of bovine inhibin. In conclusion, the present study has revealed that the bovine ovary secretes considerable quantities of monomeric inhibin alpha subunit. The unexpected presence of this material in peripheral blood is likely to hinder attempts to obtain physiologically relevant data on circulating levels of inhibin in cattle using conventional radioimmunoassays.