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題 名 | 麩胱甘冴轉移酶對四氯異苯腈的代謝=Metabolism of Chlorothalonil by Glutathione-S-transferase |
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作 者 | 周正平; 李國欽; 蔣啟玲; | 書刊名 | 植物保護學會會刊 |
卷 期 | 41:2 1999.06[民88.06] |
頁 次 | 頁107-118 |
分類號 | 433.85 |
關鍵詞 | 麩胱甘冴轉移酶; 四氯異苯腈; 大腸桿菌; Glutathione-s-transferases; Chlorothalonil; Escherichia coli; LC/Mass; |
語 文 | 中文(Chinese) |
中文摘要 | 殺菌劑四氯異苯�繻陞堳e本省推薦使用於作物病害防治上常用之農藥。由於四氯異苯�籉b生物體內,受麩胱甘�鬋鉦濂t(GST)的催化與麩胱甘�鞳]glutathione, GSH)形成GSH-共軛衍生物,而開始一連串的代謝反應,最後形成的含硫物質會抑制粒腺體的呼吸作用,導致細胞的死亡,故引起學者廣泛的研究。本實驗之目的,即在建立以化學法及酵素法合成代謝產物-四氯異苯�禲籿SH衍生物,並探討分離自人體內,非病原的大腸桿菌中之GST,對四氯異苯�穭壯@用。發現酵素法可直接得到代謝衍生物,化學合成則因副反應同時會產生其它的副反應產物,將目標物以HPLC淨化後,再經LC/MS鑑定分子量,而得到與期望之衍生物相吻合的質譜圖,證明代謝衍生物可經由化學及酵素法合成。以超高速離心,硫酸銨沈澱區分及親和管柱分離等步驟,分離來自大腸桿菌的GST,以40 ul (1.0 ug/ml)酵素液催化四氯異苯�禲]39 uM,約10 ppm)及GSH 1.0 mM反應24小時後,所得之代謝衍生物量較對照組(GST去活化)為高,表示非病原性大腸桿菌中的GST會代謝四氯異苯�籈峖沁SH-衍生物。 |
英文摘要 | Clorothalonil is an important broad spectrum contact fungicide that has been widely used to control diseases of plants for about 25 years. One toxicological issue that continues to cause regulatory concern by EPA of U.S.A. is that chronic dietary administration of chlorothalonil to rodents induces a series of nonneoplastic histopathological changes in the epithelial cells of the proximal tubule of the kidney and the squamous epithelium of the forestomach. The mechanisms of the histopathlogical changes are initiated by the glutathione S-transferase (GSTs') enzymatic reaction to produce the glutathion-conjugated compounds, which are metabolized to cytotoxic thiols by cysteine conjugated β-lyase pathway. Our study is to identify the glutathionyl conjugates of chlorothalonil 1-3 componunds by using LC/MS and to isolate the GSTs from the non-pathogenic Escherichia coli for assaying the GSH-conjugated compounds formed by this enzyme. The results show that the high purity of chlorothalonil-GSH conjugates 1-3 could be obtained by enzymatical methods. And the molecular weights of the glutathionyl conjugates of chlorothalonil 1-3 are agreeable with LC/MS determined, after clean-up by HPLC.E.coli E1 was used as sources of GST. The bacterial cells were grown at 37 ℃ in 1..5 liters of LB broth, and were harvested and washed twice with buffer. After the cells were broken by ultra sonication, the cell homogenate was centrifuged at 100,000xg for 1hr. The supernatant was used as crude enzyme extract. The enzyme was purified stepwisely by ammonium sulfate fractionation and GSH-affinity chromatography. 40 ul of partially purified GST (1.0 ug/ml) was reacted with .0 mM GSH and 39 uM chlorothalonil for 24h. The amount of GSH-conjugates of chlorothalonil produced was higher than the control (1.0 mM GSH and 39 uM chlorothalonil only). |
本系統中英文摘要資訊取自各篇刊載內容。