廖秀娟臺灣大學：生物環境系統工程學研究所曾月怡Tseng, Yuen-YiYuen-YiTseng2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/55965砷是一種毒性物質且是已知的人類致癌物質。在原核生物中，砷的解毒機制有較詳盡的研究，當類金屬進入細胞時，ars operon中的ArsA蛋白受到激發且水解ATP，使三價砷和三價銻化合物轉移到ArsB蛋白運送出細胞。其中，人類中ArsA同源體也被證實具有水解ATP的活性，然而卻只受到三價砷的激發，而酵母菌中的ArsA同源體則是不受任何類金屬的激發。因此，探討Caenorhabditis elegans（C. elegans）中ArsA同源體的功能，能更加了解C. elegans中砷的解毒途徑和獲得演化上間接的證據。 本研究利用生物資訊方法預測出C. elegans中具有兩個ArsA蛋白的同源體，我們發現藉由RNA干擾技術抑制Ce-ArsA蛋白的產生，將會造成C. elegans對三價砷和三價銻的抵抗性降低，顯示Ce-ArsA蛋白扮演了重要抵抗類金屬的角色。此外，Ce-arsA-1上游基因的分析和轉殖C. elegans的實驗，顯示當C. elegans處於不同壓力的情況時，Ce-ArsA-1蛋白的表達量也會相對明顯地增加，推測這些壓力因子活化了Ce-arsA-1的轉錄機制。 本研究利用原核生物系統大量表達和純化Ce-ArsA-1蛋白，並利用呈色法檢測純化過的Ce-ArsA-1蛋白。結果顯示Ce-ArsA-1蛋白的確具有ATPase活性，並且可被三價砷和三價銻分別激發2.2和4.8倍，此實驗結果與原核中ArsA蛋白功能較為相似，並且由研究結果證實C. elegans中Ce-ArsA-1蛋白與原核中ArsA蛋白皆屬於相同類型的ATP水解蛋白。Arsenic is a potent toxin and carcinogen. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid operon-encoded efflux systems. Two Caenorhabditis elegans genes, Ce-arsA-1 and Ce-arsA-2, that are homologous to the Escherichia coli (E. coli) arsA genes were identified and characterized in this study. The level of Ce-arsA-1 transcription was significantly affected by heat, metalloids and heavy metal exposure. When Ce-arsA-1 expression was inhibited using RNA interference (RNAi), lower resistance of arsenite and antimonite were observed. This suggests that Ce-ArsA-1 is required for arsenite and antimonite tolerance in C. elegans. We expressed and purified Ce-ArsA-1 protein, a nematode homologue of arsA encoding the ATPase component of E. coli arsenite/antimonite transporter. Purified MBP-Ce-ArsA-1 fusion protein was biochemically characterized, and its properties were compared with those of E. coli ArsA. The MBP-Ce-ArsA-1 exhibited a basal level of ATPase activity in the absence of arsenite or antimonite. Antimonite induced a 4.8 fold stimulation of ATPase activity, which was related to an increase in Vmax; arsenite induced 2.2 fold stimulation of ATPase activity. The results indicate that Ce-ArsA-1 is a distinct arsenite and antimonite-stimulated ATPase belonging to the same superfamily of ATPases represented by the E. coli ArsA protein.致謝 I 中文摘要 II ABSTRACT III Table of Contents IV List of Tables VI List of Figures VII Abbreviations IX CHAPTER 1 INTRODUCTION 1 1.1 Arsenic (As) 1 1.2 Antimony (Sb) 2 1.3 Detoxification Systems of Arsenic and Antimony in Bacteria 3 1.4 ArsA in Prokaryotes 7 1.5 ArsA in Eukaryotes 8 1.6 Caenorhabditis elegans (C. elegans) 11 1.7 Purpose of Study 13 CHAPTER 2 MATERIALS AND METHODS 15 2.1 Chemicals 15 2.2 Strains, Clone, and Culture Condition 15 2.3 RNA Interference (RNAi) 15 2.4 Growth and Isolation of C. elegans 17 2.5 RNA Isolation 17 2.6 Reverse Transcription and Polymerase Chain Reaction (RT-PCR) 18 2.7 Effect of Transgenic C. elegans 18 2.8 Construction of Recombinant Ce-ArsA-1 for Protein Expression 20 2.9 Purification of Maltose Tagged Ce-ArsA-1 Protein 20 2.10 ATPase Activity Assays 21 2.11 Fluorescence Microscope 22 2.12 Statistical Analysis 22 CHAPTER 3 RESULTS 24 3.1 Structure and Organization of The Ce-ArsA-1 24 3.2 RNA Interference 29 3.3 Effect of Various Stressors on Ce-ArsA-1 Transcription 37 3.4 Recombinant Ce-ArsA-1 Protein Expression and Purification 40 3.4.1 Overexpression of Ce-ArsA-1 protein 40 3.4.2 Purification of the Ce-ArsA-1 protein 44 3.5 ATPase Activity Assay of Ce-ArsA-1 44 3.5.1 Effect of enzyme concentration and temperature on ATPase activity 44 3.5.2 Effect of Metal ions on ATPase Activity of Ce-ArsA-1 Protein 46 3.5.3 Affinity of the Ce-ArsA-1 Protein for substrates 50 CHAPTER 4 DISCUSSIONS 53 4.1 Identification and Characterization of the Nematodes Homologue of the Bacterial arsA Gene 53 4.2 Functions of Ce-ArsA-1 in C. elegans 54 4.3 How As(III) and Sb(III) are Exported in C. elegans? 58 CHAPTER 5 CONCLUSION 62 REFERENCE 63 APPENDIX 703430151 bytesapplication/pdfen-US砷銻Caenorhabditis elegansCe-ArsAATPase活性ArseniteAntimoniteATPase activitythesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/55965/1/ntu-95-R93622049-1.pdf