Activity was determined after 1, 2 or 12 mo after purification with the 4-MUP technique

Activity was determined after 1, 2 or 12 mo after purification with the 4-MUP technique. enzyme in recognition kits. Appearance from the enzyme as a recombinant protein provided a solution to this problem. For this purpose, several strategies have been followed. We evaluated the activity, specificity and stability of the human protein phosphatase 2A catalytic subunit expressed in insect larvae and showed that this expression system can be a reliable source of high quantities of stable enzyme. responsible for amnesic shellfish poisoning, species of dinoflagellates from the genera Alexandrium, Pyrodinium and Gymnodinium that cause the paralytic shellfish poisoning, Karenia responsible for the neurotoxic shellfish poisoning, Dinophysis and Prorocentrum responsible for the diarrheic shellfish poisoning (DSP) and Gambierdiscus responsible for the ciguatera fish poisoning. In freshwater, the most ZM39923 important HABs are caused by certain species of cyanobacteria from the genera Anabaena, Microcystis, and Apyanizomenon.10 The toxins, small non-peptides, are some of the most powerful natural substances known.11 In the marine and freshwater systems, humans and animals can get exposed to HA toxins by eating contaminated fish or shellfish, drinking contaminated water, inhaling contaminated aerosol, or by contacting contaminated water. With increasing worldwide seafood consumption and trade, as well as international tourism, these diseases are expanding beyond their traditional geographic boundaries producing serious consequences on ZM39923 human health and industry. It was estimated that at least US$ 449,291,987 were spent on dealing with the known HABs from 1987 to 1992 in public health, commercial fishery, recreation/tourism and monitoring/management in the US alone. 12 Diarrheic and Hepatotoxic Toxins Among the previously mentioned organisms, Dinophysis, Prorocentrum, Microcystis and Planktothrix, produce toxins [okadaic acid (OA), dinophysis toxin-1 and -2 (DTX-1 and -2) and microcystins] that ZM39923 are potent inhibitors of protein phosphatases 1, 2A and 2B (PP1, PP2A and PP2B). Of the three phosphatases, PP2A is the most strongly inhibited.13,14 The toxins from these organisms, are responsible of the diarrheic shellfish poisoning (DSP) and can produce liver damage in humans and animals.15,16 They are globally widespread and their blooms are predicted to increase, as a consequence of natural or anthropogenic eutrophication (enhanced phytoplankton growth due excess supply of nutrients).11 Diarrheic toxins and microcystins pose an important threat for human and animal health, and are also responsible for important fish and shellfish industry loses. As previously mentioned, the blooms of toxin producing organisms is predicted to increase, so the development of rapid, sensitive, and inexpensive methods to monitor the DSP toxins and microcystins occurrence in water and contaminated shellfish is needed, in order to manage the health and economic risk posed by these toxins. PP2A as a Tool for Toxin Detection Based on the PP2A inhibitory capacity of OA, DTXs and microcystin, initially assays for determining OA shell-fish contamination were developed using enzymes purified from animal tissues.17,18 These methods have not been widely used due to fluctuations in enzyme quality. One of the sources of these fluctuations is the enzyme quaternary structure that can change during purification, and differs between different tissues. The PP2A (Fig.?1) is a trimmer consisting of a 36 kDa catalytic subunit (PP2AC), and two regulatory subunits, A and ZM39923 CSH1 B. The core enzyme consists of the catalytic subunit and the regulatory subunit A (PP2AD). Two isoforms are known of subunits A (A and A) and C (C and C). Subunit B associate to the core enzyme and regulates the enzyme localization and specific activities, and several isoforms have been identified.19 PP2A has been purified in both, dimeric and trimeric forms,20,21 while purification procedures have been applied to obtain trimeric PP2A without the presence of PP2AD.22 This indicates that depending the purification procedure, different forms of the enzyme can be obtained. Besides this, there are other draw backs in using PP2A purified from animal tissues; when purified from muscle, kilos of tissue are needed and the purification process involves several chromatographic actions (4 to 9, depending the purification procedure),22 making the process expensive and time consuming, even more if large quantities of enzyme are needed to use in multiple assays. These problems, observed when purifying PP2A from animal tissues gives an idea of the fluctuations in enzymatic stability and composition that makes at least complicated, the use of this type of PP2A in assays for toxin detection. In order for an enzyme to be used in a microplate assay, high purity, ZM39923 stability, and sensitivity are essential. So, to satisfy these needs, recombinant PP2A has been produced in different hosts..