Liquid chromatography-linked protein phosphatase bioassay; a highly sensitive marine bioscreen for okadaic acid and related diarrhetic shellfish toxins.
Okadaic acid and dinophysistoxin-1 were resolved by liquid chromatography, then identified and quantitated by specific inhibition of both protein phosphatase-1 and -2A (PP1/PP2A) catalytic subunits in a 32P-phosphorylase a phosphatase radioassay. Based on the IC50 for PP2A inhibition (0.2 nM), the procedure has a detection sensitivity of less than 10 pg okadaic acid. Confirmative identification by PP1 inhibition (IC50 = 19 nM) requires 500 pg okadaic acid. Analyses of methanolic extracts from control, "okadaic acid spiked" and suspected diarrhetic mussels showed the bioscreen to be accurate, reproducible and identified okadaic acid/dinophysistoxin-1 in Canadian shellfish for the first time. In addition, a protein phosphatase inhibitor distinct from okadaic acid/dinophysistoxin-1 was identified in diarrhetic mussels with a potency equivalent to 900 ng okadaic acid/g digestive tract. Protein phosphatase inhibition probably underlies the biological activity of okadaic acid as a diarrhetic shellfish toxin and tumour promoter (Cohen, P., Holmes, C. F. B. and Tsukitani, Y. (1990), TIBS 15, 98-102). The liquid chromatography-linked protein phosphatase bioscreen should therefore facilitate identification of novel toxins comprising diarrhetic profiles in infested shellfish.
Holmes
C F
CF
National Research Council Canada, Biotechnology Research Institute, Montreal.
D000818:Animals; D001681:Biological Assay; D049872:Bivalvia / Q000032:analysis*; D002853:Chromatography, Liquid; D003967:Diarrhea / Q000139:chemically induced*; D004988:Ethers, Cyclic / Q000032:analysis; D008387:Marine Toxins / Q000032:analysis*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors; D054645:Protein Phosphatase 1; D012680:Sensitivity and Specificity
D004988:Ethers, Cyclic; D008387:Marine Toxins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1