http://scholars.ntou.edu.tw/handle/123456789/15585
標題: | Fluorescence versus Radioactivity for Assaying Antifungal Compound Inhibited Yeast 1,3-β-glucan Synthase Activity | 作者: | Yuan-Tih Ko Wei-Yi Cheng |
關鍵字: | 1,3-β-glucan synthase;aniline blue;antifungal;yeast;echinocandin | 公開日期: | 1-六月-2005 | 出版社: | 衛生福利部食品藥物管理署 | 卷: | 13 | 期: | 2 | 起(迄)頁: | 184 - 191 | 來源出版物: | 藥物食品分析 | 摘要: | Yeast membrane (1,3)-β-glucan synthase (GS; EC 2.4.1.34) activity was assayed by a fluorescent dye-binding method and a conventional radioactivity method to compare its in vitro inhibition by the echinocandin-class antifungal compound. In the fluorescence method, GS-containing microsomal plasma membrane was first reacted with UDP-glucose substrate to form the 1,3-β-glucan product. The product was then solubilized and bound specifically with aniline blue to form a 1,3-β-glucan-fluorochrome dye complex for reporting the GS activity. The effect of each component in the GS assay buffer on fluorescence was examined. A calibration curve was constructed using yeast glucan as standard. In addition, the GS activities using the same amount of 1 μg/μL microsomal concentration in the two assays were compared. The radioactivity assay showed 38.3 μM UDP-glucose incorporation and corresponded to the formation of equivalent 262 μM yeast glucan in the fluorescent assay. It indicated a possible underestimation of 1,3-β-glucan products in the radioactivity assay or an overestimation of the products in the fluorescence assay. However, pneumocandin A0 exhibited an IC50 value of 1.25 μM in the fluorescent assay closely comparable with that of 1 μM in the radioactivity assay. In summary, both the fluorescence and radioactivity tests generated similar data and pneumocandin inhibited GS. Possible limitations in the radioactivity assay were also discussed. |
URI: | http://scholars.ntou.edu.tw/handle/123456789/15585 | DOI: | 10.6227/jfda |
顯示於: | 食品科學系 |
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