Molés Gregorio, Gómez Ana, Rocha Ana, Carrillo Manuel, Zanuy Silvia
Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Científicas, 12595 Torre la Sal, Ribera de Cabanes s/n, Castellón, Spain.
Gen Comp Endocrinol. 2008 Aug;158(1):68-76. doi: 10.1016/j.ygcen.2008.05.005. Epub 2008 May 16.
Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40-85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50-1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0-50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein alpha (GPalpha) and 13.6 kDa for FSHbeta subunits. After separation by SDS-PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPalpha and FSHbeta). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17beta (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPalpha and FSHbeta), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.
从海鲈（欧洲鲈）垂体中纯化出促卵泡激素（FSH），并对其生化和生物学特性进行了研究。通过乙醇提取沉淀法（40 - 85%）纯化海鲈FSH（sbsFSH），随后使用FPLC系统，在LKB Ultropac TSK - DEAE柱上采用碳酸氢铵线性梯度（50 - 1000 mM）进行阴离子交换色谱，以及在RESOURCE 15RPC柱上采用乙腈线性梯度（0 - 50%）进行反相色谱。通过质谱估计，纯化后的sbsFSH二聚体分子量为28.5 kDa，糖蛋白α（GPalpha）为12.6 kDa，FSHβ亚基为13.6 kDa。在还原条件下经SDS - PAGE分离后，完整的sbsFSH解离为各自的亚基（GPalpha和FSHβ）。通过免疫检测和N端氨基酸测序确认了亚基的一致性。用N - 糖苷酶F进行去糖基化处理降低了两个亚基的分子量。完整的sbsFSH以剂量依赖方式稳定激活在细胞系HEK 293中稳定表达的海鲈FSH受体。纯化后的sbsFSH通过以剂量和时间依赖方式刺激海鲈卵巢释放雌二醇 - 17β（E2）以及体外培养的睾丸组织释放睾酮（T）和11 - 酮睾酮（11KT），表现出促性腺活性。这些结果表明，纯化后的sbsFSH是一种异源二聚体激素，由两个不同的糖蛋白亚基（GPalpha和FSHβ）组成，并具有通过以特定方式刺激其受体以及促进性腺组织片段释放类固醇来判断的生物学活性。
