大鳞大马哈鱼垂体促甲状腺素和促性腺激素分离纯化的研究

本文报道应用ConA-Sepharose 4B.亲和层析、凝胶过滤层析、离子交换层析及免疫亲和层析等技术,首次从大鳞大马哈鱼垂体中分离纯化了促甲状腺素(sTSH)和促性腺激素(sGTH)。在TSH生物测定中,纯化的。sTSH制品具有明显促进虹鳟幼鱼甲状腺体内分泌甲状腺素(T4)的生物活性,而sGTH无此活性。在GTH生物测定中,制备的sGTH具有显著诱导虹鳟卵母细胞体外培养成熟(GVBD)及分泌17α、20β-二氢孕酮的能力,而sTSH无此活性。HPLC表明sTSH和sGTH的分子量分别为27500和38000道尔顿。作者用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦检测了激素的纯度及等电点,用RIA或ELISA方法测定了几种垂体激素在sTSH终产物中的污染程度。

SEPARATION AND PURIFICATION OF PITUITARY THYROTROPIN AND GONADOTROPIN OF CHINOOK SALMON (ONCORHYNCHUS TSCHAWYTSCHA)

The isolation and purification of thyrotropin (TSH) and gonadotropin (GTH) from the pituitary of Chinook Salmon were carried out by: (1). Alkaline extraction of acetone dried pituitary powder; (2). affinity chromtography of the pituitary extracts on Con A-Sepharose 4B; (3) filtration chromatography of glycoprotein on Ultro gel AcA 54; (4). cation exchange chromatography of fractions with thyrotropic and gonadotropic activities on carboxyme hyl (CM) Bio-gel A and (5). immunoaffinity chromatography of TSH active fraction on immobili zed anti-beta SGTH antibody Sepharose 4B. The TSH molecule purified was homogenous in SDS-PAGE and exhibited high thyrotropic activity since this preparation stimulated thyroxine (T4) release in vivo in the homologous TSH bioassay. Gonadotropin (GTH), Somatotropin (GH) and prolactin (PRL) contaminations in the purified sTSH final preparation were very low. In an in vitro GTH bioassay, the sTSH exhibited no gonadotropic activity whereas the purified sGTH preparation exhibited high steroidogenic activities and maturational activities. Molecular weights were estimated by HPLC to be 27500 and 38000 dalton for sTSH and sGTH, respectively Isoelectric focusing showed that both sTSH and sGTH consisted of multiple components which differed in isoelectric points ranging between 5.4-7.0 for sTSH and between 3.5-5.5 for sGTH.