Isolation of Inhibin α-Subunit Precursor Proteins from Bovine Follicular Fluid
Isolation of Inhibin α-Subunit Precursor Proteins from Bovine Follicular Fluid
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从牛卵泡液中分离抑制素 α-亚基前体蛋白
DOI：
10.1210/endo-125-4-2141
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发表时间：
1989
期刊：
影响因子：
4.8
通讯作者：
D. Kretser
中科院分区：
文献类型：
--
作者：
D. Robertson;M. Giacometti;L. M. Foulds;J. Lahnstein;N. Goss;M. Hearn;D. Kretser
从牛卵泡液中分离出两种蛋白质，其结构特征与在α-亚基前体序列中鉴定的肽序列相似。从牛卵泡液中分离纯化出一个具有高水平免疫活性的牛卵泡素侧馏分，该侧馏分通过一系列程序进行分离，包括三嗪染料亲和层析和苯基-琼脂糖凝胶层析，Sephadex G-100凝胶渗透层析，反相HPLC和制备聚丙烯酰胺凝胶电泳。在还原和非还原条件下鉴定的两种蛋白质中的第一种具有25- 26 K的分子量，并且其NH 2-末端序列与43 K α-亚基的NH 2-末端序列相同，并且与牛31 K α-亚基相比，在α-亚基体外生物测定或RIA中显示出最小活性（小于2%活性）。这些数据表明，这种蛋白质是牛胰蛋白酶α-亚基（称为α N-亚基）的α 1-166序列，最有可能在胰蛋白酶α-亚基前体或参与58 K至31 K胰蛋白酶转化的43 K α-亚基加工后释放。另一种蛋白质（命名为pro-alpha C-亚基）在非还原条件下的分子量为27 K，在还原条件下的分子量为20 K和6 K。它在体外生物测定中无活性，但在拟南芥素RIA中具有高度反应性，并且具有与拟南芥素α-亚基前体的原序列和20 K α-亚基序列相同的NH 2-末端。这些结果表明，pro-alpha C是一个二硫键连接的结构，并可能代表在二聚化的α-和β-亚基，以形成reflubin的中间体，而α N-亚基可能是一个蛋白水解产物的α-亚基前体或58 K reflubin。
Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.