Intracellular localization of tetrodotoxins in pufferfish liver

采用三种不同毒性河豚（Takifugu pardalis， T.porphyreus, t.m vermicularis snyderi）研究了河豚肝脏中蛇毒毒素（TTXs）的细胞内定位。肝脏在组织匀浆器中切碎并匀浆，匀浆器中有3体积20mM maps - koh缓冲液（pH7.2），其中含有60mM KCl， 180mM蔗糖，1mM EGTA和10muM苯基甲基氟磺酸（PMSF），转速为200 rpm，持续30分钟。去除组织碎片后，用离心法将匀浆分离成五部分（血细胞、细胞核、线粒体、微粒体和细胞质）。用0.1%醋酸在沸水中加热10 min，提取各馏分中的毒素。小鼠实验表明，5个部位的提取物均有致死毒性，但不同部位的毒素含量有显著差异。大约80%的毒素总量在细胞质中被回收，1-10%在血细胞、细胞核和线粒体中被回收，约1%在微粒体中被回收，无论种类和肝脏的毒性水平如何。这些结果表明，毒素广泛分布于肝细胞的脏器中。高效液相色谱（HPLC）、气相色谱-质谱联用、液相色谱-快速原子轰击质谱联用分析表明，TTX是各组分中主要的毒素成分。此外，即使不经醋酸处理，其胞浆在小鼠生物测定中也显示出毒性，HPLC也发现其毒素为TTX。结果表明，TTX在细胞质中首先以游离形式存在，不与其他物质结合。

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.