Grootenhuis A J, Timmerman M A, Hordijk P L, de Jong F H
Department of Biochemistry, Erasmus University Rotterdam, The Netherlands.
Mol Cell Endocrinol. 1990 Mar 26;70(1):109-16. doi: 10.1016/0303-7207(90)90064-f.
Conditioned medium of cultured Sertoli cells from 21-day-old rats was used as starting material for the isolation of inhibin. Inhibin activity was monitored by the dose dependent suppression of the follicle-stimulating hormone release of cultured rat pituitary cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the highly purified inhibin preparation revealed a 32 kDa protein after silver staining, which could be separated in subunits of 18 kDa and 12 kDa after reduction. Western blot analysis with an antibody recognizing the 22 N-terminal amino acids of the alpha-subunit of 32 kDa bovine inhibin confirmed the presence of a 32 kDa inhibin molecule under non-reducing conditions, whereas an 18 kDa alpha-subunit was found after reduction. An antibody recognizing the beta-A subunit of inhibin did not yield a signal after Western blotting. N-terminal amino acid sequence analysis of two highly purified preparations of inhibin obtained using different methods yielded the sequence predicted for a 32 kDa alpha beta-B dimer on basis of cDNA nucleotide sequence. This result is in agreement with the large excess of beta-B over beta-A mRNA in the rat testis.
将21日龄大鼠培养的支持细胞的条件培养基用作分离抑制素的起始材料。通过培养的大鼠垂体细胞促卵泡激素释放的剂量依赖性抑制来监测抑制素活性。对高度纯化的抑制素制剂进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，银染后显示出一条32 kDa的蛋白质条带，还原后可分离为18 kDa和12 kDa的亚基。用识别32 kDa牛抑制素α亚基22个N端氨基酸的抗体进行蛋白质印迹分析，证实在非还原条件下存在32 kDa的抑制素分子，而还原后发现有一个18 kDa的α亚基。识别抑制素β-A亚基的抗体在蛋白质印迹后未产生信号。对使用不同方法获得的两种高度纯化的抑制素制剂进行N端氨基酸序列分析，得到了基于cDNA核苷酸序列预测的32 kDaαβ-B二聚体的序列。该结果与大鼠睾丸中β-B mRNA大大过量于β-A mRNA的情况一致。
