| 中文摘要 |
酵母菌1,3-β-葡聚醣合成瓷（EC 2.4.1.34）的活性利用螢光性染劑結合的方法與傳統式的放射性方法來測量，並用於被echinocandin類的抗黴藥化合物作用的抑制性比較；在螢光法中，含有葡聚醣合成瓷的胞漿膜微粒體首先與基質UDP-glucose反應形成葡聚醣產物，接著產物會被溶解，並與苯氨藍進行特異性結合，形成1,3-β-葡聚醣-螢光子-複合物來報導酵素活性。酵素活性測試液成份對螢光的影響也進行測試，檢量線則以酵母菌葡聚醣為標準品來建立。葡聚醣合成瓷的活性以這兩種方法進行比較，並使用1 μg/μL相同濃度的胞漿膜微粒體反應，結果，酵素活性以放射法測量，會得到38.3μM UDP-glucose基質濃度參與合成反應，相較於螢光法，卻相當於得到262μM葡聚醣產物的生成，顯示出利用放射性方法會有低估生成物的現象，或螢光法會有高估生成物的現象；雖然如此，抗黴藥pneumocandin A0的作用以螢光法呈現出其抑制的IC50值為1.25 μM，卻和以放射性法所得抑制值1μM相近。本研究總結，放射性與螢光性兩種方法可以檢測出被echinocandin類抗黴藥作用的相近抑制值，對於放射性方法中的可能限制性也有所討論。" |
| 英文摘要 |
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. |
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