微囊藻毒檢測方法與毒理機制的探究

Studies on the Detection and Toxicology of Microcystins

陳逸民(Yi-Min Chen)

指導教授 ： 黃穰 ； 周宏農

國立臺灣大學/理學院/海洋研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.02050

摘要

首先以現有的微囊藻株為材料，分別測試其對於小白鼠、豐年蝦的毒性，以及其對於蛋白質磷酸水解酵素PP-1c的抑制活性後，分析此三種活性表現之關連性。由對於PP-1c具有抑制活性的藻株，亦對兩種生物皆具明顯毒性的初步結果，推測微囊藻毒對於兩種生物的毒性，可能均與其抑制蛋白質磷酸水解酵素的活性有關。在以自行設計的矽膠固相萃取法處理粗萃樣品後，將使三種活性分析數值間一致性更佳的結果，說明本法的使用，有助於改善上述分析方法對於微囊藻毒分析的精確性。其對於微囊藻樣品內各種微囊藻毒的回收率，另經後續MALDI-TOF及HPLC的分析而獲肯定。
後續以由上述藻株中所純化出之各種微囊藻毒純質，分別測定其對於小白鼠的急性毒性、PP-1c及PP-2Ac兩種蛋白質磷酸水解酵素的抑制活性，以及其與PP-1c形成共價產物之動力學。藉由微囊藻毒結構與活性關連性數值的分析，推測出微囊藻毒的第三氨基酸MeAsp，在抑制PP-2Ac的活性表現中所扮演的角色，並不若其於抑制PP-1c來的重要，同時得知微囊藻毒的急性毒性和PP-2Ac抑制的關連，遠高於其對PP-1c的抑制。在以MALDI-TOF分析MCYST-LR與PP-1c共價鍵形成動力學的研究中，得知在30oC的反應條件下，每小時僅有低於5 %的PP-1c會形成共價鍵，反應速率極為緩慢。此說明共價鍵的形成應為毒素以其他非共價鍵的分子間作用力與PP-1c結合，同時抑制其活性後再出現的事件，與毒素的抑制活性間並無直接的關連。

關鍵字

微囊藻毒

並列摘要

Strains of Microcystis have been cultured for their toxicity analysis, and their toxins were isolated for their specific toxicity tests. Mouse and Artemia toxicity assays were applied for the various strains and their respective LD50 and LC50 that were compared with the IC50 of protein phosphatase inhibition assay after converted to the toxin content of MCYST-LR equivalent. Strains that do not possess any PP1 inhibitory activity show no toxicity to either mice or brine shrimp larvae. Ranking of the animal toxicities coincided well with enzyme inhibitory activities. A solid phase extraction procedure was developed for the removal of interfering agents in Artemia assay and proved to be very effective. This SPE pretreatment in conjugation with the following assays can be applied for the MCYST screening in field cyanobacterial samples or in algal food supplements. MALDI-TOF and HPLC metabolomic analysis showed the SPE good in preserving the toxins and removing contaminants.
In the further study a series of microcystins were tested on the mouse toxicity, protein phosphatase inhibition activities of PP-1 and PP-2A from native or recombinant sources, and the covalent-bonding kinetics with recombinant PP-1c. Structure-Activity-Relationship studies showed mouse toxicities of different microcystins were in better correlation with their inhibitory activities against PP2Ac than PP1c. Different microcystins have different inhibitory activities on different enzyme sources, indicating the different interaction in molecular level. The covalent-bonding kinetics obtained from MALDI-TOF assays showed that less than 5% of total PP-1c covalent-bonding with MCYST-LR per hour under 30oC, revealing the slow reacting nature of covalent-bonding formation between PP-1c and microcystin-LR.

並列關鍵字

microcystin

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