Grünhagen H H, Dahl G, Reiter P
Biochim Biophys Acta. 1981 Apr 6;642(2):267-85. doi: 10.1016/0005-2736(81)90445-4.
A tetrodotoxin receptor-rich preparation of membrane fragments from the electric organ of Electrophorus electricus is described. The specific binding of neurotoxins and freeze-fracture electron microscopy are used as tools to identify and to characterize membrane fractions. Freeze-fracture electron micrographs of the electric organ demonstrate a high density of membrane particles in the extrasynaptic regions. Density gradient fractions show a broad distribution of [3H]tetrodotoxin, [3H]saxitoxin and 125I-labelled bungarotoxin binding in the range of 1.04--1.15 g/ml sucrose densities, with specific neurotoxin binding up to approx. 5 pmol/mg protein. Carrier-free column electrophoresis of density gradient fractions yields a subfraction with tetrodotoxin and alpha-neurotoxin binding up to 30 pmol/mg protein. The major part of the membrane fragments forms vesicles, which are separated by lectin chromatography into an outside-out and inside-out population. The latter represents at least 50% of the material of a density gradient fraction. For the association of tetrodotoxin, a bimolecular kinetic constant kf greater than or equal to 3.10(5) M-1.s-1 is determined. The dissociation constant is k'b = 2.5.10(-2)s-1. These data are in agreement with a thermodynamic dissociation constant of Kd = 20 nM as determined earlier for E. electricus membrane fragments by equilibrium methods (Grünhagen, H.H., Rack, M., Stämpfli, R., Fasold, H. and Reiter, P. (1981) Arch. Biochem. Biophys. 206, in the press). However, these association kinetics of tetrodotoxin binding in vitro are significantly different from kinetics determined electrophysiologically in Rana (Wagner, H.H. and Ulbricht, W. (1975) Pflügers Arch. 359, 297--315) or Xenopus (Schwarz, J.R., Ulbricht, W. and Wagner, H.H. (1973) J. Physiol. 233, 167--194).
本文描述了一种从电鳗（Electrophorus electricus）电器官中制备的富含河豚毒素受体的膜碎片制剂。使用神经毒素的特异性结合和冷冻断裂电子显微镜作为工具来鉴定和表征膜组分。电器官的冷冻断裂电子显微照片显示突触外区域的膜颗粒密度很高。密度梯度分级显示，[3H]河豚毒素、[3H]石房蛤毒素和125I标记的银环蛇毒素在1.04 - 1.15 g/ml蔗糖密度范围内有广泛分布，特异性神经毒素结合量高达约5 pmol/mg蛋白质。密度梯度分级的无载体柱电泳产生一个亚组分，其河豚毒素和α-神经毒素结合量高达30 pmol/mg蛋白质。膜碎片的主要部分形成囊泡，通过凝集素色谱法将其分离为外翻和内翻群体。后者占密度梯度分级材料的至少50%。对于河豚毒素的结合，确定了双分子动力学常数kf大于或等于3.10(5) M-1.s-1。解离常数为k'b = 2.5.10(-2)s-1。这些数据与之前通过平衡方法（Grünhagen, H.H., Rack, M., Stämpfli, R., Fasold, H. and Reiter, P. (1981) Arch. Biochem. Biophys. 206, in the press）测定的电鳗膜碎片的热力学解离常数Kd = 20 nM一致。然而，河豚毒素结合的这些体外结合动力学与在蛙（Wagner, H.H. and Ulbricht, W. (1975) Pflügers Arch. 359, 297 - 315）或非洲爪蟾（Schwarz, J.R., Ulbricht, W. and Wagner, H.H. (1973) J. Physiol. 233, 167 - 194）中通过电生理学测定的动力学有显著差异。
