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| Title: | 利用蛋白質體學研究含吡咯環抗生素之抑菌作用 |
| Other Titles: | Chemical proteomics of antibiotics containing pyrrole rings |
| Authors: | 陳盈廷;Chen, Ying-ting |
| Contributors: | 淡江大學生命科學研究所碩士班
鄭建中;Cheng, Chien-chung |
| Keywords: | 蛋白質體學;偏端黴素;紡錘菌素;Proteomics;distamycin;netropsin |
| Date: | 2005 |
| Issue Date: | 2010-01-11 02:31:28 (UTC+8) |
| Abstract: | 蛋白質體學是二十一世紀對新藥開發的重要利器。利用蛋白質體學技術標的出受抗生素影響而發生變化的蛋白質，能提供較完整的藥物以及其生物活性之間關係。本論文利用兩種已知與DNA小溝鍵結之抗生素，紡錘菌素(netropsin)與偏端黴素(distamycin)進行測試。前者在結構上少一個甲基吡咯基團，但多一個正電荷的胍基。紡錘菌素對d(GGTATACC)2鍵結能力為1×105 M-1，是偏端黴素的0.5倍。但在本實驗中，發現紡錘菌素抑制大腸桿菌BL21生長能力較強，所需的濃度低於偏端黴素八倍。基於藥物與DNA作用後，會影響蛋白質生成，導致生物活性的改變。不同濃度之紡錘菌素（7, 11, 15 µM）及偏端黴素（60, 90, 120 µM），對細菌進行培養17小時後，經破菌，丙酮或硫酸銨沈澱後進行透析，得到總體蛋白質，再經一維的等電聚焦電泳，及二維聚丙醯凝膠分離後，比對蛋白質之間的差異。再利用基質輔助雷射質譜儀鑑定出蛋白質之分子量，即可找出所對應的可能代謝途徑。為了解抗生素對蛋白質生成的調控影響，在不同時間下(3，5，8，17小時)，亦分別對總體蛋白質變化進行分析。結果發現：紡錘菌素和偏端黴素在DNA雖有相似的影響，但因結構的差異性，導致所表現出的蛋白質不同，而改變細菌成長活性。因此本論文研究結果，對藥物的結構及生物活性之間的相對關係，將提供一個展新的思考方向。
Proteomic is an important tool for new drug discovery in the 21st century. The understanding about the influence of total proteins in drug provides the more information about the relationship between the structure of drug and biological activity. Two kinds of antibiotics, netropsin and distamycin, were examined the correlation in this thesis. These two antibiotics are known to bind at the DNA minor groove, the former structure has one less methylprrrole, this being replaced with guanidimium group carring positive charge. The binding affinities to netropsin and [d(GGTATACC)]2 are determined to be 1×105 M-1 that is half to that of distamycin. However, the inhibition concentration of netropsin in the bacterial growth is eight-times to that of distamycin. DNA modification with drug will influence its protein distribution resulting in the different of biological activity. The use of proteomic technology is capable of monitoring the variation of total protein. In our laboratory, different concentration of netropsin (7, 11, 15 µM) and distamycin (60, 90, 120 µM) was allowed to incubate with bacterial in the medium for 17 hours, respectively. Isolation and purification by acetone or ammonium sulfate precipitation, and dialysis give total proteins prior to the separation by 1-D IEF and 2-D SDS-PAGE. Comparison of the spots isolated from 2-D gel shows the different protein distribution. Determination of molecular weight of each spot by MALDI mass spectrograph provides a reasonable metabolic pathway for these antibiotics. In order to understand the kinetic data for the protein formation, total proteins was obtained for different reaction time periods of 3, 5, 8, 17 hours. It suggests that netropsin and distamycin undergo the different metabolic pathway even though they have similar components in chemical structure.These results will offer a new strategy in drug development and have great potential for overcoming drug-resistance. |
| Appears in Collections: | [Graduate Institue of Life Sciences] Thesis
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