鯽魚卵細胞硫氫蛋白酶抑制劑之純化、特性分析及基因選殖

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標題:	鯽魚卵細胞硫氫蛋白酶抑制劑之純化、特性分析及基因選殖 Purification, characterization and Genetic Cloning of Cystatin from Crucian Carp (Carassius Auratus) Oocyte
作者:	劉健安 Liu chien-an
貢獻者:	曾鑫順嘉南藥理科技大學：生物科技研究所
關鍵字:	特性分析純化硫氫蛋白酶抑制劑卵細胞鯽魚 Crucian carp oocyte cysteine proteinase inhibi
日期:	2007
上傳時間:	2008-12-03 11:16:53 (UTC+8)
摘要:	二種純化自鯽魚卵細胞的硫氫蛋白酶抑制劑分別定名為 CST-I 和 CST-II，純化步驟包括酸處理、CM-Sepharose FF 以及 Sephacryl S-100 HR 管柱層析。硫氫蛋白酶抑制劑 CST-I 與 CST-II 純化倍率為 98 與 112 倍，而回收率分別為 22 % 和 20 %。二者分子量經 SDS-PAGE 電泳分析測定，CST-I 為 14 kDa，而 CST-II 則為 9.2 kDa；未添加 β-mercaptoethanol，CST-I 及 CST-II 分子量皆為 14 kDa。更進一步，將 CST-I 以質譜儀進行分析，得到其精確分子量為 12258 Da。這二種抑制劑的 pH 安定性為 2.0-11.0，且能耐受 4-70 oC 恒溫 1 小時。蛋白酶活性測定與膠體活性染色分析，顯示 CST-I 及 CST-II 均能有效抑制木瓜蛋白酶，但無法抑制組織蛋白酶 B 與胰蛋白酶，這說明了此二種抑制劑為硫氫蛋白酶專一抑制劑。CST-I 與 CST-II 的 N 端胺基酸序列分別為 AGIPGGLVDA 與 GIPGGLVDAD，並與鯉魚 cystatin 之胺基酸序列極為接近，相似度高達 90%。 Two cysteine proteinase inhibitors, designated as CST-I and -II, were purified from the oocyte of crucian carp. The purification included acidification,CM-Sepharose FF, and Sephacryl S-100 HR chromatographs. The purification fold and recovery of CST-I were 98, and 22%, while those of CST-II were 112 and 20% respectively. The molecular masses (M) of CST-I and II estimated on SDS-PAGE were 14 kDa and 9.2 kDa. However, both inhibitors were shown to be about 14 kDa on -me-free SDS-PAGE. Furthermore, the CST-I analyzed on MALDI-TOF spectrometer had a M of 12258 Da. No significant loss in the inhibitory activity within pH 2.0 ~ 11.0, 40 oC and 1 hr of incubation at 4~70 oC. Both cystatins could effectively inhibit papain, but not trypsin and cathepsin B. The N-terminal sequences of CST-I and -II were identical, which were determined to be AGIPGGLVDA and GIPGGLVDAD. They shared a 90% identity of amino acid sequences cystatin of with common carp (Cyprinus carpio).
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