Good T E, Weber P S, Ireland J L, Pulaski J, Padmanabhan V, Schneyer A L, Lambert-Messerlian G, Ghosh B R, Miller W L, Groome N
Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, East Lansing, Michigan 48824, USA.
Biol Reprod. 1995 Dec;53(6):1478-88. doi: 10.1095/biolreprod53.6.1478.
A combination of immunoaffinity chromatography, SDS-PAGE, and electroelution was used to simultaneously isolate 0.36-4.65 mg of nine different molecular forms of inhibin (pro alpha C-29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 58, 77, 88, 110, and > 160 kDa) from 0.675 L of bovine follicular fluid (bFF). Each inhibin form, except pro alpha C, cross-reacted with inhibin alpha C 1-26-and beta A 82-114-subunit-directed antibodies during immunoblot analysis. Pro alpha C cross-reacted only with alpha-subunit antibodies. The inhibin forms consisted of 22-, 29-, 49-, or 58-kDa alpha subunits and 17- or 58-kDa beta subunits. During cultures of ovine pituitary cells, a 5-ng/ml dose of each inhibin form (except pro alpha C) suppressed basal accumulation of FSH 30% to 50% but increased GnRH-induced LH release 40% to 248%. The various inhibin forms cross-reacted in parallel fashion with standard curves generated during homologous and heterologous RIAs but with markedly different relative immunopotencies. In the RIAs, pro alpha cross-reacted 3- to 18-fold more than the fully processed inhibin form. The fully processed and the seven different large forms of inhibin cross-reacted with different relative immunopotencies in a two-site dimer-specific ELISA. We concluded that 1) a combination of immunoaffinity extraction, SDS-PAGE, and electroelution simultaneously isolated relatively large amounts of highly enriched preparations of nine different molecular forms of immunologically and biologically active inhibin from bFF; 2) eight different dimeric forms of bovine inhibin may regulate both basal FSH and GnRH-induced LH secretion by the pituitary gland, and 3) eight or nine different molecular forms of inhibin cross-react with different relative immunopotencies in the two-site dimer-specific assay or RIAs.
采用免疫亲和色谱、SDS - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和电洗脱相结合的方法，从0.675升牛卵泡液（bFF）中同时分离出0.36 - 4.65毫克9种不同分子形式的抑制素（前αC - 29 kDa；完全加工的34 kDa；以及大抑制素形式49、53、58、77、88、110和>160 kDa）。除前αC外，每种抑制素形式在免疫印迹分析中均与抑制素αC 1 - 26和βA 82 - 114亚基导向抗体发生交叉反应。前αC仅与α亚基抗体发生交叉反应。抑制素形式由22 kDa、29 kDa、49 kDa或58 kDa的α亚基和17 kDa或58 kDa的β亚基组成。在绵羊垂体细胞培养过程中，每种抑制素形式（除前αC外）5纳克/毫升的剂量可使基础促卵泡激素（FSH）积累量抑制30%至50%，但使促性腺激素释放激素（GnRH）诱导的促黄体生成素（LH）释放增加40%至248%。各种抑制素形式在同源和异源放射免疫分析（RIA）过程中与标准曲线以平行方式发生交叉反应，但相对免疫活性明显不同。在RIA中，前α的交叉反应比完全加工的抑制素形式高3至18倍。在双位点二聚体特异性酶联免疫吸附测定（ELISA）中，完全加工的抑制素和7种不同的大抑制素形式以不同的相对免疫活性发生交叉反应。我们得出以下结论：1）免疫亲和提取、SDS - PAGE和电洗脱相结合的方法可从bFF中同时分离出相对大量的、高度富集的9种不同分子形式的具有免疫活性和生物活性的抑制素制剂；2）8种不同的牛抑制素二聚体形式可能调节垂体的基础FSH和GnRH诱导的LH分泌；3）8或9种不同分子形式的抑制素在双位点二聚体特异性测定或RIA中以不同的相对免疫活性发生交叉反应。
