Bandyopadhyay S, Bhattacharya S
Department of Zoology, Visva-Bharati University, West Bengal, India.
Gen Comp Endocrinol. 1993 May;90(2):192-204. doi: 10.1006/gcen.1993.1074.
Thyroid stimulating hormone (TSH) was isolated and purified from the pituitaries of Indian major carp, Cirrhinus mrigala, with the help of a sensitive in vitro bioassay system, based on incubating isolated thyroid follicles from murrel, Channa gachua. In this assay, addition of test material containing TSH increased thyroxine (T4) release into the medium which was then measured by radioimmunoassay (RIA). TSH activity was eluted as one major peak on Sephadex G-100 gel filtration (peak I), which also contained gonadotropin (GtH) and extraneous proteins as contaminants. GtH was monitored by RIA. Chromatography of peak I of gel filtration on concanavalin A-Sepharose was useful in harvesting glycoprotein hormones; adsorbed (Con A-II) material showed strong TSH activity and low GtH content. To separate TSH from GtH completely, immunoaffinity chromatography was used; more than a 200-fold purification was achieved. Polyacrylamide gel electrophoresis of carp TSH (cTSH) showed a single band, indicating it to be a homogenous protein. The molecular weight (MW) of cTSH was estimated by Sephadex G-100 gel filtration to be 42,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cTSH revealed two dissimilar subunits, alpha and beta, and MW of alpha was 21,000 Da while that of beta was 24,000 Da. When tested in similar doses, cTSH released more T4 from murrel thyroid follicle than bovine TSH (bTSH). cTSH stimulated the T4 release from rat and goat thyroid but its activity was less than that of bTSH. Radiolabeled cTSH (125I-cTSH) bound specifically to a murrel thyroid follicular plasma membrane preparation, and Scatchard analysis showed the Kd to be 0.17 x 10(-10) M and the maximum binding (Bmax) to be 15.2 fmol/mg protein. 125I-cTSH also binds to the goat thyroid plasma membrane preparation with a Kd = 0.23 x 10(-10) M and a Bmax = 9.8 fmol/mg protein. Findings indicate that carp pituitary thyrotropin is approximately similar in size to that of vertebrates, its biological activity is not restricted only to fish but is also observed in rat and goat, and it has a receptor in teleost and goat thyroid.
借助一种灵敏的体外生物测定系统，从印度主要鲤鱼——印度野鲮（Cirrhinus mrigala）的垂体中分离并纯化出促甲状腺激素（TSH）。该生物测定系统基于孵育来自攀鲈（Channa gachua）的分离甲状腺滤泡。在这个测定中，添加含有TSH的测试物质会增加甲状腺素（T4）释放到培养基中，然后通过放射免疫分析（RIA）进行测量。TSH活性在葡聚糖凝胶G - 100凝胶过滤中作为一个主要峰被洗脱出来（峰I），该峰还含有促性腺激素（GtH）和作为污染物的外来蛋白质。通过RIA监测GtH。在伴刀豆球蛋白A - 琼脂糖上对凝胶过滤的峰I进行色谱分析有助于收获糖蛋白激素；吸附的（伴刀豆球蛋白A - II）物质显示出很强的TSH活性和低GtH含量。为了将TSH与GtH完全分离，使用了免疫亲和色谱法；实现了超过200倍的纯化。鲤鱼TSH（cTSH）的聚丙烯酰胺凝胶电泳显示为一条带，表明它是一种同质蛋白质。通过葡聚糖凝胶G - 100凝胶过滤估计cTSH的分子量（MW）为42,000道尔顿。cTSH的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示出两个不同的亚基，α和β，α的MW为21,000道尔顿，β的MW为24,000道尔顿。当以相似剂量进行测试时，cTSH从攀鲈甲状腺滤泡中释放的T4比牛TSH（bTSH）更多。cTSH刺激大鼠和山羊甲状腺释放T4，但其活性低于bTSH。放射性标记的cTSH（125I - cTSH）特异性结合到攀鲈甲状腺滤泡质膜制剂上，Scatchard分析显示解离常数（Kd）为0.17×10⁻¹⁰ M，最大结合量（Bmax）为15.2 fmol/mg蛋白质。125I - cTSH也以Kd = 0.23×10⁻¹⁰ M和Bmax = 9.8 fmol/mg蛋白质的结合特性结合到山羊甲状腺质膜制剂上。研究结果表明，鲤鱼垂体促甲状腺激素的大小与脊椎动物的大致相似，其生物活性不仅限于鱼类，在大鼠和山羊中也可观察到，并且它在硬骨鱼和山羊甲状腺中有受体。
