Origin of tetrodotoxin and toxification mechanism in tetrodotoxin-bearing animals

点击翻译按钮获取中文摘要

Horseshoe crabs are often eaten by the people in Thailand, and food poisonings by ingesting them, especially their eggs have sporadically occurred. Horseshoe crabs are primitive arthropods, and only three genera and four species are surviving now in the world. Carcinoscorpius rotundicauda, Tachypleus gigas and T.tridentatus are Asean horseshoe crabs and Limulus polyphemus is an American species. The former two species inhabit in Thailand. The causative species was C.rotundicauda in all the cases reported. The responsible toxin is essentially composed of tetrodotoxin (TTX) and related substances, along with paralytic shellfish toxins (PSP) as minor components. The mechanism of intoxication of the crabs is unhnown. Therefore we examined the torelance of the crabs against TTX as well as PSP, because there is a tendency that an animal with high tolerance to toxins may contain the toxins or have the capability to accumulate them. As to TTX, T.gigas and L.polyphemus exibited almost the same considerable high tolerance, with the minimum lethal dose (MLD) of 50-150 MU/20g of body weight. While C.rotundicauda showed a very high resistance that was about 40 times stronger than the others. As to PSP, C.rotundicauda and L.polyphemus exibited almost the same considerable high tolerance, with MLD of 100-200 MU/20g of body weight.A tetrodotoxin binding protein has been purified from the plasma of the puffer fish kusafugu, Takifugu niphobles, through DBAB-cellulose treatment, ammonium sulfate fractionation, Sephadex gel filtrations, and Sephacryl S-200 and Cellulofine A-500 column chromatography. Final purification by I-IPLC on a BK 0-3000 SL column yielded a protein which showed only a single protein peak. The molecular weight of the protein was estimated to be 116,000 and 96,000 by SDS-PAGE and mass spectrometry, respectively. The amino-terminal amino acid sequence of the purified protein was determined to be Ala-Pro-Ser-Pro-? -? -? -His-? -Leu-The-Lys-Rro-Val-.