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

Abstract

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