A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins

被引：34

作者：

Ikehara, Tsuyoshi ^{[1
]}

Imamura, Shihoko ^{[1
]}

Oshiro, Naomasa ^{[2
]}

Ikehara, Satsuki ^{[1
]}

Shinjo, Fukiko ^{[1
]}

Yasumoto, Takeshi ^{[3
]}

机构：

[1] Trop Technol Ctr Ltd, Okinawa 9042234, Japan

[2] Okinawa Prefectural Inst Hlth & Environm, Okinawa 9011202, Japan

[3] JST, Okinawa CREATE, Okinawa Hlth Biotechnol Res Dev Ctr, Okinawa 9042234, Japan

来源：

TOXICON | 2008年 / 51卷 / 08期

关键词：

ELISA; microcystins; protein phosphatase 2A (PP2A) inhibition assay; recombinant protein;

D O I：

10.1016/j.toxicon.2008.03.003

中图分类号：

R9 [药学];

学科分类号：

1007 ;

摘要：

Worldwide blooms of toxic cyanobacteria (blue-green algae) commonly occur in freshwater, often in drinking water sources, necessitating routine monitoring of water quality. Microcystin-LR and related cyanobacterial toxins strongly inhibit protein phosphatase 2A (PP2A) and are therefore assayable by measuring the extent of PP2A inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant PP2A (rPP2Ac) expressed with a baculovirus system for use in a microplate microcystin assay. Five microcystin analogs, microcystin-LR, -RR, -YR, -LF, and -LW, and nodularin strongly inhibited rPP2Ac activity with IC50 values of 0.048, 0.072, 0.147, 0.096, 0 114, and 0.54 nM, respectively. Microcystin-LR in a water sample could be assayed from 0.005 to 5 ng/ml. The assay could detect the toxin at a far lower level than required by the World Health Organization for regulation of microcystin-LIZ or its equivalent (1 mu g/L). Pretreatment or concentration of water samples with low toxin concentrations was not necessary. The microplate assay using rPP2Ac was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method and a cytotoxicity assay. The genetically engineered rPP2Ac was more stable than a commercially available dimeric enzyme, producing accurate and reproducible results. Our results confirm that the rPP2Ac we prepared is an excellent tool for detecting and quantifying microcystins in water. (c) 2008 Elsevier Ltd. All rights reserved.

引用

页码：1368 / 1373

页数：6

共 18 条

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