碩士 / 國立臺灣大學 / 漁業科學研究所 / 89 / 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 PP1 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 32.3 pmol/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 0.01-0.20 ng/ml (r2 >0.99). Its sensitivity is higher than HPLC 1000 times.
The PP1 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 (M.TN-1, M.KS-1 and C.TN-1).
Compared with PP1 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.
| Identifer | oai:union.ndltd.org:TW/089NTU00451001 |
| Date | January 2000 |
| Creators | Jian-Iu Bai, 白健瑜 |
| Contributors | Hong-Nong Chou, 周宏農 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 84 |
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