DEVELOPMENT OF A PROTEIN PHOSPHATASE-BASED ASSAY FOR THE DETECTION OFPHOSPHATASE INHIBITORS IN CRUDE WHOLE-CELL AND ANIMAL EXTRACTS

Diarrhetic shellfish poisoning (DSP) is a serious and globally widespr ead phytoplankton-related seafood illness. Although DSP is rarely life -threatening, it causes incapacitating diarrhea and vomiting with no k nown medical treatments. In addition, phytoplankton producing DSP toxi ns have been identified in temperate coastal waters worldwide, and the ir numbers may be increasing as a result of coastal eutrophication. Th e toxic effects of the major DSP toxins, okadaic acid and dinophysisto xin-1 (35-methylokadaic acid), appear to originate from their inhibito ry activity against a family of structurally related serine/threonine protein phosphatases (PSPases). In particular, the inhibition of essen tial PSPases (e.g. PP1 and PP2A) has catastrophic consequences in most eukaryotic cells. Exploiting the potent inhibitory property of the DS P toxins, we have developed an enzyme-based assay (PP2A assay) capable of detecting both okadaic acid and dinophysistoxin-1 in nanogram amou nts. The assay employs purified PP2A, which has an extremely high affi nity for both DSP toxins. This provides the PP2A assay with a level of sensitivity comparable to, or surpassing, that of most monoclonal ant ibody probes. To evaluate the PP2A assay as a means of detecting conta minated shellfish, a series of spike recovery experiments was conducte d. The findings from these studies suggest that the PP2A assay has the potential for development into a rapid and relatively simple method f or detecting PSPase inhibitors in crude extracts produced from shellfi sh. Copyright (C) 1996 Elsevier Science Ltd