okadaic-acid has been researched along with 4-methylumbelliferyl-phosphate* in 2 studies
2 other study(ies) available for okadaic-acid and 4-methylumbelliferyl-phosphate
Article | Year |
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Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).
Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability. Topics: Acridines; Animals; Bivalvia; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Fluorescein; Fluorometry; Hymecromone; Indicators and Reagents; Marine Toxins; Okadaic Acid; Phosphoprotein Phosphatases; Reproducibility of Results; Shellfish; Substrate Specificity | 2000 |
A fluorescent microplate assay for diarrheic shellfish toxins.
A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples. Topics: Animals; Diarrhea; Fluoresceins; Fluorescent Dyes; Fluorometry; Humans; Hymecromone; Marine Toxins; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Protein Phosphatase 2; Reproducibility of Results; Sensitivity and Specificity; Shellfish; Shellfish Poisoning; Substrate Specificity | 1997 |