蛋白磷酸酵素抑制性分析於微囊藻株之毒性檢測研究

Abstract

微囊藻毒（Microcystins）是一群具有肝臟毒性的環狀勝?類化合物，同時也是抑制蛋白磷酸?l(protein phosphatase-l, PP-l）及蛋白磷酸?2A(protein phosphatase-2A,PP-2A）的腫瘤促進因數（tumor promoter)，其主要的產毒生物為藍綠藻的微囊藻屬（Microcystis spp.)。微囊藻分佈於溫帶、亞熱帶及熱帶地區，常見於台灣各水域，包括淡水、半淡鹹水養殖池及水庫等，容易形成藻華，不但造成養殖池生物的死亡，亦污染水庫而引起飲用水方面的安全問題。所以快速而靈敏的微囊藻毒檢測分析，一直是研究者追求的目標。 本研究利用蛋白磷酸?1在對-硝基酚磷酸(p-nitrophenyl phosphate, p-NPP）上之脫磷酸反應，對不同濃度微囊藻純毒MCYST-LR及多株包括產毒及不產毒之不同微囊藻株進行毒性檢測。研究工作首先以酵素動力學之相關參數確定酵素反應之條件，並獲得本研究所使用蛋白磷酸?1酵素之Km=5.lmM、Vmax=32.3pmole/min，而在操作所使用的濃度下，反應60分鐘後的受質消耗率為0.035%，並依該條件進行毒素抑制性的檢視。 微囊藻純毒MCYST-LR的酵素活性抑制試驗顯示直線抑制藻毒濃度範圍為0.01-0.20ng/ml(r2>0.99)，具有20-80％的抑制活性，其靈敏度比高效液相層析高出1000倍，足以適用於WHO所限制飲用水含微囊藻毒量1ng/ml。 以酵素活性抑制試驗法檢測產毒及不產毒藻株，結果顯示在所有含毒藻株中，當每毫升含有相當於250ng或更低乾藻重的草出物，就可檢測到對PPI即具有80％以上的抑制活性；而在無毒藻株，則需要高達每毫升2.3-22.7mg才能顯示出對PPI 3％的抑制力。同時，依據各藻株之半抑制濃度IC50及抑制曲線之斜率，可正確區別出各藻株間之毒性差異，在所有測試的含毒藻株中，可分為包括藻株M.TY-l,MTN-4的強抑制組與包括藻株M.TN-2, M.TN-3, M.CY-1的弱抑制組而 M.TY-2藻株介於兩組之間。此結果與毒藻萃出物在小白鼠與豐年蝦之生物毒性分析的結果，具有相同的趨勢。 本研究証明，以酵素活性抑制試驗法在微囊藻之毒性分析上，具準確性、高靈敏度及時效性上的優點，同時也可顯示蛋白磷酸酵素活性的抑制與小白鼠或豐年蝦毒性的相關性。

Microcystins (MCYSTs) are the group of hepatotoxic cyclic peptides compounds and the inhibitor of protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A). They are also considered as tumor promoters. Some of the blue-green algae species, especially M. areuginosa, produce MCYSTs, commonly found in the temperater, subtropical and tropical zones. When they form a algal bloom caused the death of livestock and wild fowls. They present a real threat to public health. Therefore, to establish fast and sensitivity detect method become the most important issue of researcher. In this research, we used the authentic MCYST-LR and 9 cyanobacterial strains extract in establishing the optimal conditions for enzyme inhibition assay of PP 1 on the conversion of p-NPP (a phophated precursor) to a UV-absorption product, p-NP. Inhibitory activities were measured by the amount of p-NP produced in the presence of toxins divided by the amount produced in control. The catalysis rate of phosphate group of p-NPP followed Michaelis-Menten kinetics. At PP1 activity 0.025 unit/well and react 60 min, Vmax was 32.3pmol/min, Km was 5.1mM and the substrate consumption was 0.035%. At the same condition the detection range of MCTST-LR from the standard curve was 0.01-0.20ng/ml (r2>0.99). Its sensitivity is higher than HPLC 1000 times. The PP 1 inhibition assay also has ability to distinguish from toxic and non-toxic cyanobacterial strains. Compared IC50 value and slope of inhibition curve can exactly apply to relative toxicity in analysis of different M. aeruginosa strains. According to IC50 and slope of all assayed strains distinguish into four domains: strong toxicity group (M.TY-1 and M.TN-4), middle toxicity group (M.TY-2), weak toxicity group (M.TN-2, M.TN-3 and M.CY-1) and non-toxic group (MTN-1, M.KS-1 and C.TN-1). Compared with PP 1 inhibition assay with the mouse bioassay and Artenmia bioassay, the potency of PP1 inhibitory activity, mouse toxicity and shrimp toxicity are the same. It is proved that PP1 inhibition assay can take the place of bioassay.