大頭鰱性腺刺激素次單元之性狀

Abstract

摘要 本實驗的目的是分離大頭鰱(Hypophthalmichthys nobilis; Bighead Carp)腦下腺性腺刺激素及次單元。 大頭鰱腦下腺粉末以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％)沈澱醣蛋白。所得之醣蛋白以Sephadex G-100分離，得到三個部分，其中第二部分(BH G2)具有最強的性腺刺激素活性。 本實驗發展以HPLC分離大頭鰱性腺刺激素次單元之系統。Column為Nucleosil 7μ C18之reverse phase column；溶劑系統包括溶劑A(水：氰甲烷：三氟醋酸＝77.5:22.5:0.07體積比)及溶劑B(水：氰甲烷：三氟醋酸＝68:32:0.07體積比)；滌流條件為溶劑B對溶劑A作非線性梯度的濃度增加，於30分鐘時，溶劑B達85％。BH G2在此系統中展開為五個部分，C1?C5；其中C1及C5為最主要部分。經過各次單元重組結合後之生物活性測定，氨基酸組成分析及N-末端部分氨基酸順序分析，我們認為C1,C2，C3為大頭鰱性腺刺激素的α次單元，C4及C5為β次單元。 α次單元(C1)之氨基酸組成中含高量的Asp(10.71%),Cys(9.41%)及val(9.09%)，而β%單元(CS)則含有高量的Glu(10.56%),Pro(9.54%),Asp(10.80%)，及Val(11.00%)。經由SDS-PAGE的鑑定，α次單元的分子量為17,400 daltons，β次單元為26,000daltons。

Abstract The present study was aimed to isolate the subunits of bighead carp (Hypophthalmichthys nobilis) gonadotropin and to characterize their chemical and biological properties. Bighead carp pituitary glands were extracted with 40% ethanol—6% ammonium acetate solution, pH5.l at 4°C, and precipitated with ethanol (final concentration 80%). The crude glycoprotein was then chromatographed by Sephadex G-l00. Three fractions were obtained, in which the second fractron (BH G2) possessed the highest biological GTH activity. In this study, isolation of subunits of bighead carp GTH was performed by high performance liquid chromatography (HPLC). Reverse phase column of Nucleosil 7μ Cl8 was used; the solvent system included solvent A (water: acetonitrile: trifluroacetic acid=77.5:22.5:0.07 v/v) and solvent B (water: acetonitrile: trifluroacetic acid=68:32:0.07 v/v); concentration of solvent B was eluted from 0 to 85% non-linearly within 30min. There were five fractions obtained from this HPLC system. They were designed as C1 to C5 according to their retention time, with C1 and C5 being the two major fractions. From the data of amino acid composition, N-terminal partial sequence and the biological GTH activity of recombinants, we inferred that C1, C2 and C3 belonged to the α subunit whereas C4 and C5 belonged to the β subunit. The amino acid composition of the α subunit (C1) was rich in Asp (10.71%), Cys(9.41%) and Val(9.09%), while the β subunit (C5) was rich in Glu(10.86%), Pro(9.84%), Asp(10.80%)and Val(11.00%). From the result of SDS-PAGE, we estimated that the molecular weights of α and β subunits were 17,400 daltons and 26,000 daltons, respectively.