Intracellular localization of tetrodotoxins in pufferfish liver

以3种不同毒性的东方鲀（Takifugu pardalis，T. porphyreus，T. vermicularis snyderi）为材料，研究了河豚鱼肝脏中河豚毒素（TTXs）的细胞内定位。将肝脏切碎并在组织匀浆器中以200 rpm的速度用3体积的含有60 mM KCl、180 mM蔗糖、1 mM EGTA和10 μ M苯基甲磺酰氟（PMSF）的20 mM MOPS-KOH缓冲液（pH 7.2）匀浆30分钟。核辐射，线粒体fr.，微粒体Fr.，和胞质溶胶fr.）用0.1%乙酸在沸水中加热10 min，从各组分中提取毒素。5个组分的提取物对小鼠均有致死毒性，但各组分的毒素量有显著差异。约80%的总毒素量在胞质溶胶中回收，血细胞fr中各占1 - 10%，核FR，和线粒体FR，约1%在微粒体FR中，而不考虑物种和肝脏的毒性水平。结果表明，毒素广泛分布于肝细胞各器官中，经高效液相色谱（HPLC）、气相色谱-质谱（GC-MS）和液相色谱-快原子轰击质谱（LC-MS）分析，毒素成分主要为TTX。此外，胞质溶胶fr.，经HPLC测定，其毒素为TTX，说明TTX在胞液中以游离形式存在，不与其它物质结合。

Intracellular localization of terodotoxins (TTXs) in pufferfish liver was investigated using three species of puffers (Takifugu pardalis, T.porphyreus, and T.vermicularis snyderi) with a variety of toxicity. Livers were minced and homogenized in a tissue homogenizer with 3 volumes of 20mM MOPS-KOH buffer (pH7.2) containing 60mM KCl, 180mM sucrose, 1mM EGTA,and 10muM phenylmethanesulfony fluoride (PMSF) at 200 rpm for 30 min. After removal of tissue debris, the homogenate was separated into five fractions (blood cell fr., nuclear fr., mitochondrial fr., microsomal fr., and cytosol fr.) by a centrifugation method.Toxins were extracted from each fraction by heating with 0.1% acetic acid in boiling water for 10 min. All the extracts from the five fractions showed lethal toxicity on mouse assay, although the toxin amount markedly varied from fraction to fraction. About 80% of the total toxin amounts was recovered in the cytosol fr., 1-10% each in the blood cell fr., the nuclear fr., and the mitochondrial fr., and about 1% in the microsomal fr., regardless of species and toxicity levels of livers. These results indicate that the toxins are widely distributed in organcella in liver cells.Analyzes by high performance liquid chromatogarphy (HPLC), gaschromatography-mass spectrometry, and liquid chromatography-fast atom bombardment mass spectrometry demonstrated that TTX was the major toxin principle in each fraction. In addition, the cytosol fr., even without the treatment with acetic acid, showed toxicity on mouse bioassay and the toxin in it was also found to be TTX by HPLC.It was concluded that TTX in the cytosol fr.exists in a free form, without binding to other substances.