鯉魚腦下腺性腺刺激素次單元之分離及特性研究

Abstract

本實驗的目的是分離鯉魚腦下腺性腺刺激素的次單元並對各次單元進行化學特性及生物活性的探討。 鯉魚腦下腺以40％乙醇，6％醋酸銨PH5.1的溶液於4℃下萃取，再以乙醇（最後濃度80%）沉澱醣蛋白。所得之醣蛋白以Sephadex G-25膠質管柱層析去除?類，之後進行DEAE-Cellulose離子交換管柱層析，收集120?300mM碳酸氫銨所條洗的部分，稱為DE300。DE300再以Sephadex G-100膠質管柱層析，得到五個部分，其中第二部分(FII)具有最強的性腺刺激素活性，此部分即本實驗所稱的鯉魚性腺刺激素。 本實驗以高壓液相層析分離鯉魚性腺刺激素各次單元，操作條件如下：分離管柱為Chemcopak7 ODS-H (4.6×250mm)之逆相管，溶劑系統包括溶劑A(水：氰甲烷?三氟醋酸＝80:20:0.04體積比)及溶劑B(水：甲醇?氰甲烷?三氟醋酸＝62.5:12.5:25:0.04體積比)；滌洗條件為?溶劑B對溶劑A作線性梯度的濃度增加，於28分鐘時，溶劑B達50％。 cGTH(FII)在此系統中展開為9個部分，依其滯留時間的短長，分稱F1?F9。經過氨基酸組成分析，部分N-末端氨基酸順序解析，以及各次單元重組結合生物活性測定，我們認為F1?F5屬於性腺刺激素一個次單元而F6?F9屬於另一次單元。F1?F9的醣類組成也被測定。 鯉魚性腺刺激素與羊黃體成長激素的次單元重組的實驗發現，在四種可能的組合中，只有羊LHα與鯉F8的重組體對促進鯉魚睪丸組織雄性素的產生具有協同作用，而其他三種組合則無，故我們認為F1?F5相當於哺乳類的α次單元而F6?F9則對應於β次單元。

The present study was aimed to isolate the subunits of carp gonadotropin (cGTH) and then to characterize their. chemical and biological properties. Carp pituitary glands were extracted with 40% ethanol-6% ammonium acetate, PH 5.1 at 4℃. The crude glycoprotein was desalted by Sephadex G-25 gel filtration and then chromatographed by DEAE-cellulose. Carp GTH was eluted by 120-300 mM NH4HCO3. This fraction was then chromato-graphed by Sephadex G-100. Five fractions were obtained, in which fraction II (FII) possessed the highest GTH activity. FII was used as intact cGTH throughout this study. In this study, isolation of subunit of cGTH was performed by high pressure liquid chromatography (HPLC). Operation was performed as follows: column, Chemcopak 7 ODS-H (4.6×250 mm); solvent system, solvent A (water: acetonitrile: trifluroacetic acid=80:20:0.04, v/v) and solvent B (water: methanol: acetonitrile: trifluroacetic acid 62.5:12.5:25:0.04, v/v); elution, concentration of solvent B increased to 50% linearly within 28 min. There were 9 fractions obtained from HPLC. They were designated as F1 to F9 according to their retention time (from short to long duration). From the data of amino acid composition, partial N-terminal sequence and the GTH activity of recombinants, we inferred that F1-F5 belonged to one subunit whereas F6-F9 belonged to another subunit. In this study, carbohydrate composition was also measured. In this study, recombination of the subunits of ovine LH and cGTH was also performed. Among the 4 possible combinations, only the recombinant of ovine LH -carp F8 possessed the synergistic effect on stimulation of carp testis to produce androgens whereas others did not. Therefore we assumed that F1-F5 and F6-F9 should correspond to mammalian α and β subunits, respectively.