PP2A Inhibition Assay Using Recombinant Enzyme for Rapid Detection of Okadaic Acid and Its Analogs in Shellfish

Ikehara, Tsuyoshi; Imamura, Shihoko; Yoshino, Atsushi; Yasumoto, Takeshi

doi:10.3390/toxins2010195

Cited by 26 publications

(27 citation statements)

References 20 publications

(31 reference statements)

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“…The calibration curves obtained demonstrate that these phycotoxins can be quantified with high sensitivity and reproducibility, which despite not being within the highest sensitivities, such as those demonstrated by Ikehara [17], it has to be considered that the sample volume used in the assay is 20 μL, and knowing that the IC50 obtained for OA is 0.86 ng mL -1 , a detection test would require 16.1 picograms to be at this level. Considering the international safe limit of 160 μg of OA equivalent per one kg of shellfish meat, these values corresponds to 3200 picograms of OA for the proposed test., being more than 360 times the amount required for the IC50.…”

Section: Discussionmentioning

confidence: 87%

“…Considering the international safe limit of 160 μg of OA equivalent per one kg of shellfish meat, these values corresponds to 3200 picograms of OA for the proposed test., being more than 360 times the amount required for the IC50. There is evidence in the literature that suggests the use of the inhibitory test of PP2A as a method to detect OA and MC-LR [3,12,17,30], since this enzymatic method is more sensitive than HPLC with Fluorescence on line detection. Therefore, an ideal method for detection in samples with low levels of phycotoxins with values under the safe limits [31][32][33].…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, recent years saw the development of an enzymatic assay based on the inhibition by these phycotoxins of their specific targets, the protein phosphatases [12,17] like, for example, PP2A that undergoes a powerful enzyme-ligand interaction which totally inhibits the enzyme [4,18]. Because PP2A has a high affinity for toxins as OA and MC-LR, with a range of inhibition between 0.02-0.2 nM [6,19], the development of an enzymatic assay using PP2A allows a very sensitive and quantitative analysis [15,20].…”

Section: Introductionmentioning

confidence: 99%

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High Yield Purification of Mytilidae PP2A: An Enzyme for the Sensitive Detection of Phycotoxins

Sepúlveda¹

2019

IJBP

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Diarrheic Shellfish Poison (DSP) toxins such as Okadaic Acid (OA), Dinophisis toxin 1 and Dinophisis toxin 2, and Microcystins such as Microcystin-LR (MC-LR) are widely spread biotoxins whose molecular mechanism of action is based on the specific inhibition of protein phosphatase 2A (PP2A) with costly negative effects on public health, even at lower quantities than those established by international safe limits worldwide, due to their tumor-promoting action and hepatotoxicity. Considering these, a zero tolerance on the human consumption of these phycotoxins should be advocated. The high affinity of these phycotoxins for PP2A allows using the same enzyme as a very sensitive biosensor. Until now, PP2A has been purified following the classic protocol to produce this enzyme that is based on mammalian tissue as the source material. However, it is characterized by its low PP2A content, resulting in low yields per gram of tissue. In this study PP2A was purified from an invertebrate, a native Chilean shellfish classified as Aulacomia atra. This Mytilidae is a common endemic shellfish that evolved together with the dinoflagellate DSP phycotoxin producers in the Chilean littoral. When the tissue of these shellfish is tested for PP2A, the data show an unusually high amount of this enzyme per gram of shellfish, using these shellfish, the yield post-purification was forty times greater than those reported for other mammalian PP2A purifications. This Mytilidae PP2A is characterized by a Vmax of 7.60 pmol min-1 μg protein-1 and a Km of 32.09 mM for p-NPP. The inhibition assay resulted in an IC50 for OA and MC-LR of 0.86 ng mL-1 and 0.437 ng mL-1 , respectively. The enzymatic stability over time was evaluated, showing that the enzyme is best kept at 4°C suspended in 10% of glycerol and as such retained 80% of its enzymatic activity after 2 weeks and 60% after more than 4 weeks. Taken together, these results indicate that PP2A purified from this bivalve filter-feeder mollusk is a good candidate biosensor for the detection and

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High Yield Purification of Mytilidae PP2A: An Enzyme for the Sensitive Detection of Phycotoxins

Sepúlveda¹

2019

IJBP

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“…Recently a method for the detection of DSPs (OA and DTX 1) has been reported showing that the recombinant human PP2ACα can be used in inhibition assays for the detection of OA in shellfish. 31 The assay using the human recombinant protein was shown to be more sensitive than native PP2A. This, together with higher stability and purity make the use of recombinant PP2AC more suitable in the production of kits for the detection of OA, DTXs and microcystins.…”

Section: Resultsmentioning

confidence: 99%

Bioengineered protein phosphatase 2A

Rubiolo¹,

López-Alonso²,

Alfonso³

et al. 2013

Bioengineered

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“…The ability of OA and derivatives to inhibit protein phosphatases has been utilised in the development of sensitive assays for DSPs in shellfish [52,53,54,55]. …”

Section: Inhibition Of Protein Phosphatases By Okadaic Acid and Dementioning

confidence: 99%

Is Protein Phosphatase Inhibition Responsible for the Toxic Effects of Okadaic Acid in Animals?

Munday

2013

Toxins

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Okadaic acid (OA) and its derivatives, which are produced by dinoflagellates of the genera Prorocentrum and Dinophysis, are responsible for diarrhetic shellfish poisoning in humans. In laboratory animals, these toxins cause epithelial damage and fluid accumulation in the gastrointestinal tract, and at high doses, they cause death. These substances have also been shown to be tumour promoters, and when injected into the brains of rodents, OA induces neuronal damage reminiscent of that seen in Alzheimer’s disease. OA and certain of its derivatives are potent inhibitors of protein phosphatases, which play many roles in cellular metabolism. In 1990, it was suggested that inhibition of these enzymes was responsible for the diarrhetic effect of these toxins. It is now repeatedly stated in the literature that protein phosphatase inhibition is not only responsible for the intestinal effects of OA and derivatives, but also for their acute toxic effects, their tumour promoting activity and their neuronal toxicity. In the present review, the evidence for the involvement of protein phosphatase inhibition in the induction of the toxic effects of OA and its derivatives is examined, with the conclusion that the mechanism of toxicity of these substances requires re-evaluation.

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PP2A Inhibition Assay Using Recombinant Enzyme for Rapid Detection of Okadaic Acid and Its Analogs in Shellfish

Cited by 26 publications

References 20 publications

High Yield Purification of Mytilidae PP2A: An Enzyme for the Sensitive Detection of Phycotoxins

High Yield Purification of Mytilidae PP2A: An Enzyme for the Sensitive Detection of Phycotoxins

Bioengineered protein phosphatase 2A

Is Protein Phosphatase Inhibition Responsible for the Toxic Effects of Okadaic Acid in Animals?

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