Grünhagen H H
Institut für Physiologische Chemie der Universität des Saarlandes, D - 6650 Homburg/Saar, F.R. Germany.
Neurochem Int. 1980;2C:73-80. doi: 10.1016/0197-0186(80)90013-3.
Na-channels are prepared in membrane fragments from the electric organ of Electrophorus electricus . To identify Na-channels in biochemical preparations and to study their binding properties, tetrodotoxin is tritiated to a high specific activity of 18 000 Ci/mol. This is achieved by a defined chemical procedure from tetrodotoxin via acetylanhydrotetrodotoxin to (3)H-tetrodotoxin. (3)H-tetrodotoxin blocks Na-current in Rana esculenta nodes at a concentration of 3 nM, whereas its apparent dissociation constant for binding to Electrophorus Na-channels is K(D) = (20 +/- 10) nM. This difference may be due to a higher density of negative surface charges in nodal regions. Density gradient membrane fractions show specific binding of (3)H-tetrodotoxin up to 5 pmol/mg. Using carrier-free column electrophoresis and lectin chromatography maximal specific binding between 25 and 30 pmol/mg have been obtained. In such membrane fractions receptor densities are close to values expected for extrasynaptic regions of the excitable face of the electroplaque. (22)Na-efflux from vesicles is controlled by neurotoxins, when bulk Na- and K-concentrations are altered at the beginning of an efflux experiment. Efflux data and interaction of the vesicles with lectins suggest a high proportion of inside-out vesicles in density gradient fractions.
钠通道是从电鳗的电器官的膜片段中制备的。为了在生化制剂中鉴定钠通道并研究其结合特性，将河豚毒素氚化至18000 Ci/mol的高比活性。这是通过从河豚毒素经乙酰脱水河豚毒素到（3）H-河豚毒素的特定化学程序实现的。（3）H-河豚毒素在浓度为3 nM时可阻断食用蛙神经节中的钠电流，而其与电鳗钠通道结合的表观解离常数为K（D）=（20±10）nM。这种差异可能是由于神经节区域中负表面电荷的密度较高。密度梯度膜组分显示（3）H-河豚毒素的特异性结合高达5 pmol/mg。使用无载体柱电泳和凝集素色谱法，已获得25至30 pmol/mg之间的最大特异性结合。在这样的膜组分中，受体密度接近电板可兴奋面突触外区域预期的值。当在流出实验开始时改变大量的钠和钾浓度时，囊泡中的（22）Na流出受神经毒素控制。流出数据以及囊泡与凝集素的相互作用表明密度梯度组分中高比例的内翻囊泡。
