Hancock A D, Robertson D M, de Kretser D M
Department of Anatomy, Monash University, Clayton, Victoria, Australia.
Biol Reprod. 1992 Jan;46(1):155-61. doi: 10.1095/biolreprod46.1.155.
Stimulation of Sertoli cells from immature rats with dibutyryl cyclic (dbc) AMP resulted in a decrease in the ratio of inhibin biological (B):immunological (I) activities in vitro. To establish the basis for this decrease, culture medium from Sertoli cells stimulated with dbcAMP was fractionated by dye-affinity chromatography, reverse-phase HPLC, and preparative PAGE. Two peaks of inhibin activity were identified: a predominantly bioactive 29-kDa peak I material (B:I ratio = 5.0) and a bio-inactive, immunoactive 27-kDa peak II material (B:I ratio = 0.1). Evidence of a subunit structure was established by iodination and immunopurification using an inhibin alpha-subunit antiserum. On reduction, peak I (29-kDa) material showed bands of 19 kDa and 14 kDa, whereas peak II (27-kDa) material showed a single 20-kDa band. On the basis of HPLC retention position, molecular mass, evidence of subunit structures and their molecular masses, and inhibin in vitro bio- and immunoactivities, peak I and II materials were most likely inhibin and the alpha-subunit precursor protein pro-alpha C. Western blotting of Sertoli cell culture medium using antiserum directed against the NH2 terminal region (alpha N) of the alpha-subunit precursor also indicated the presence of 24-kDa alpha N. It is concluded that after dbcAMP stimulation, Sertoli cells produce 29-kDa inhibin and the alpha-subunit precursor proteins pro-alpha C and alpha N. The production of the alpha-subunit precursor in addition to inhibin provides an explanation for the decrease in the inhibin B:I ratio following dbcAMP stimulation of Sertoli cells in culture.
用二丁酰环磷腺苷（dbc）AMP刺激未成熟大鼠的支持细胞，导致体外抑制素生物活性（B）与免疫活性（I）的比值下降。为确定这种下降的基础，对经dbcAMP刺激的支持细胞培养基进行了染料亲和层析、反相高效液相色谱和制备性聚丙烯酰胺凝胶电泳分离。鉴定出两个抑制素活性峰：一个主要为具有生物活性的29 kDa峰I物质（B:I比值 = 5.0）和一个无生物活性、有免疫活性的27 kDa峰II物质（B:I比值 = 0.1）。通过碘化和使用抑制素α亚基抗血清进行免疫纯化确定了亚基结构的证据。还原后，峰I（29 kDa）物质显示出19 kDa和14 kDa的条带，而峰II（27 kDa）物质显示出单一的20 kDa条带。基于高效液相色谱保留位置、分子量、亚基结构及其分子量的证据以及抑制素的体外生物活性和免疫活性，峰I和峰II物质最有可能分别是抑制素和α亚基前体蛋白前αC。用针对α亚基前体NH2末端区域（αN）的抗血清对支持细胞培养基进行蛋白质免疫印迹分析也表明存在24 kDa的αN。得出的结论是，在dbcAMP刺激后，支持细胞产生29 kDa的抑制素以及α亚基前体蛋白前αC和αN。除抑制素外还产生α亚基前体，这为在培养中用dbcAMP刺激支持细胞后抑制素B:I比值下降提供了解释。
