Kistner A, Sanders D, Habermann E
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig Universität, Giessen, F.R.G.
Toxicon. 1993 Nov;31(11):1423-34. doi: 10.1016/0041-0101(93)90208-z.
The interchain disulfide bond of tetanus toxin is known to be cleaved by reduced thioredoxin and by rat brain homogenate. We now show that this bond, but not the disulfide loop in the heavy chain of the toxin, can be restored quickly and completely by oxidized thioredoxin. Oxidized glutathione was at least 100 times less potent and less specific. Reduced tetanus toxin did not measurably (KD below 50 nM) dissociate into its chains, as revealed by HPLC gel chromatography under nondenaturing conditions. Accordingly, when the reduced toxin or its recombined chains were injected into mice, general toxicity was diminished but not abolished, as compared with the native form. Inhibition of Ca(2+)-evoked [3H]noradrenaline release was assayed in cultured adrenomedullary cells after permeabilization with digitonin. Reduced two-chain tetanus toxin was as active as the isolated light chain in this system, and the action of the light chain was only slightly diminished by the addition of excess heavy chain. The results show that thioredoxin can both open and close the covalent bond between the chains of tetanus toxin, and that the reduced chains remain linked by noncovalent forces. The role of the thioredoxin system for reversible activation of tetanus toxin in vivo remains to be established.
已知破伤风毒素的链间二硫键可被还原型硫氧还蛋白和大鼠脑匀浆裂解。我们现在表明，该键而非毒素重链中的二硫环，可被氧化型硫氧还蛋白快速且完全地恢复。氧化型谷胱甘肽的效力至少低100倍且特异性较差。如在非变性条件下通过HPLC凝胶色谱法所揭示的，还原型破伤风毒素未检测到（解离常数低于50 nM）解离成其链。因此，当将还原型毒素或其重组链注射到小鼠体内时，与天然形式相比，总体毒性降低但未消除。在用洋地黄皂苷通透后，在培养的肾上腺髓质细胞中测定对Ca(2+)诱发的[3H]去甲肾上腺素释放的抑制作用。在该系统中，还原型双链破伤风毒素与分离的轻链活性相同，并且加入过量重链只会使轻链的作用略有减弱。结果表明，硫氧还蛋白既能打开又能闭合破伤风毒素链间的共价键，并且还原型链通过非共价力保持连接。硫氧还蛋白系统在体内对破伤风毒素可逆激活的作用仍有待确定。
