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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/27231

Title: Fluorescence versus Radioactivity for Assaying Antifungal Compound Inhibited Yeast 1,3-β-glucan Synthase Activity
Authors: Yuan-Tih Ko;Wei-Yi Cheng
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 1,3-β-glucan synthase;aniline blue;antifungal;yeast;echinocandin
Date: 2005-06
Issue Date: 2011-10-21T02:24:03Z
Publisher: Journal of Food and Drug Analysis
Abstract: Abstract: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.
Relation: 13(2), pp.184-191
URI: http://ntour.ntou.edu.tw/handle/987654321/27231
Appears in Collections:[食品科學系] 期刊論文

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