Govoroun M S, Huet J C, Pernollet J C, Breton B
Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, Rennes, France.
J Chromatogr B Biomed Sci Appl. 1997 Sep 26;698(1-2):35-46. doi: 10.1016/s0378-4347(97)00265-x.
A new procedure is described for the purification of gonadotropic hormones (GTHs) from the pituitary glands of vitellogenic rainbow trout. The procedure utilizes immobilized metal ion affinity chromatography (IMAC) on a column containing immobilized iminodiacetic acid (Toyopearl AF Chelate) charged with Cu2+ ions as a critical step for the efficient separation of GTH I and GTH II. Further purification of both GTH fractions on Cibacron Blue F3GA immobilized on Toyopearl was followed by HPLC size-exclusion for GTH II. The resulting electrophoretically homogeneous preparations possessed a characteristic range of biological activity in the stimulation of steroidogenesis in vitro. N-Terminal sequences of the GTH I and GTH II subunits purified using reversed-phase HPLC revealed a high level of homology with those of GTH subunits found in three other salmonid fish species. In contrast to salmon GTH II, two different alpha-subunits were observed in trout GTH II. The cross-reactivity between GTH I and GTH II was studied in radioimmunoassay using antibodies against Chinook salmon GTH II beta-subunit and rainbow trout GTH I dimer.
本文描述了一种从卵黄生成期虹鳟鱼的脑垂体中纯化促性腺激素（GTHs）的新方法。该方法利用固定化金属离子亲和色谱法（IMAC），以含有负载Cu2+离子的固定化亚氨基二乙酸（Toyopearl AF Chelate）的柱子作为高效分离促性腺激素I（GTH I）和促性腺激素II（GTH II）的关键步骤。在固定于Toyopearl的Cibacron Blue F3GA上对两种GTH组分进行进一步纯化后，再对GTH II进行高效液相色谱尺寸排阻分离。所得的电泳纯制剂在体外刺激类固醇生成方面具有一系列特征性的生物活性。使用反相高效液相色谱法纯化的GTH I和GTH II亚基的N端序列显示，与在其他三种鲑科鱼类中发现的GTH亚基具有高度同源性。与鲑鱼的GTH II不同，在虹鳟鱼的GTH II中观察到两种不同的α亚基。使用针对奇努克鲑鱼GTH IIβ亚基和虹鳟鱼GTH I二聚体的抗体，通过放射免疫分析法研究了GTH I和GTH II之间的交叉反应性。
