Isolation of nine different biologically and immunologically active molecular variants of bovine follicular inhibin.

Isolation of nine different biologically and immunologically active molecular variants of bovine follicular inhibin.
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牛卵泡抑制素九种不同的生物和免疫活性分子变体的分离。

DOI:
10.1095/biolreprod53.6.1478
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发表时间:
1995
影响因子:
3.6
通讯作者:
Groome,N
Groome,N
中科院分区:
生物学2区
文献类型:
--
作者:
Good,TE;Weber,PS;Ireland,JL;Pulaski,J;Padmanabhan,V;Schneyer,AL;Lambert-Messerlian,G;Ghosh,BR;Miller,WL;Groome,N

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采用免疫亲和层析、SDS-PAGE和电洗脱的组合,从0.675 L牛卵泡液(bFF)中同时分离0.36-4.65 mg 9种不同分子形式的Escherichin(proac-29 kDa;完全加工的34 kDa;和大Escherichin形式49、53、58、77、88、110和> 160 kDa)。在免疫印迹分析中,除proac外,每一种p53 bin形式都与p53 bin ac 1 -26-和pA 82 - 4-亚基定向抗体交叉反应。Proac仅与a-亚单位抗体交叉反应。该蛋白由22-、29-、49-或58-kDa的α亚基和17-或58-kDa的亚基组成。在绵羊垂体细胞培养过程中,5 ng/ml剂量的每种形式(除了proa)抑制FSH的基础积累30%至50%,但增加GnRH诱导的LH释放40%至248%。各种形式的交叉反应,在一个平行的方式与标准曲线产生同源和异源RIA,但具有显着不同的相对免疫效力。在RIA中,proac的交叉反应比完全加工的甜菜碱形式高3至18倍。完全处理的和七种不同的大形式的双位点二聚体特异性ELISA中的不同的相对免疫效力交叉反应。我们的结论是:1)免疫亲和提取、SDS-PAGE和电洗脱的组合同时从bFF中分离出相对大量的高度富集的九种不同分子形式的免疫学和生物学活性的Eclobin制剂; 2)牛卵泡刺激素的八种不同二聚体形式可以调节垂体的基础FSH和GnRH诱导的LH分泌,和3)在双位点二聚体特异性测定或RIA中,八种或九种不同分子形式的Escherichin与不同的相对免疫效力交叉反应。
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 (proac-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 follicularfluid (bFF). Each inhibin form, except proac, cross-reacted with inhibin ac1-26-and pA82" 4-subunitdirected antibodies during immunoblot analysis. Proac cross-reacted only with a-subunit antibodies. The inhibin forms consisted of 22-, 29-, 49-, or 58-kDa a subunits and 17-or 58-kDa subunits. During cultures of ovine pituitary cells, a 5-ng/ml dose of each inhibin form (except proa) suppressed basal accumulation of FSH 30% to 50% but increased GnRH-induced LH release 40% to 248%. The various inhibin forms cross-reacted in a parallel fashion with standard curves generated during homologous and heterologous RIAs but with markedly different relative immunopotencies. In the RIAs, proac 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.
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