Parton R G, Ockleford C D, Critchley D R
J Neurochem. 1987 Oct;49(4):1057-68. doi: 10.1111/j.1471-4159.1987.tb09994.x.
The fate of tetanus toxin bound to neuronal cells at 0 degree C was followed using an anti-toxin 125I-protein A assay. About 50% of surface-bound toxin disappeared within 5 min of warming cells to 37 degrees C. Experiments with 125I-toxin showed that much of this loss was due to dissociation of bound toxin into the medium. Some toxin was however rapidly internalised, and could be detected only by permeabilizing cells with Triton X-100 prior to assay. To investigate the mechanism of internalisation, tetanus toxin was adsorbed to colloidal gold. Toxin-gold was shown to be stable, and to recognise the same receptor(s) as free toxin. Quantitation of the distribution of toxin-gold particles bound to the cell body at 4 degrees C showed that it was concentrated in coated pits. After 5 min at 37 degrees C, toxin-gold appeared in coated vesicles, endosomes, and tubules. After 15 min, it was found largely in endosomes, and at 30 min in multivesicular bodies. The involvement of coated pits in internalisation of tetanus toxin, but not cholera toxin, was confirmed using the free toxins, anti-toxins, and protein A-gold. Toxin-gold also entered nerve terminals and axons via coated pits, accumulating in synaptic vesicles and intraaxonal uncoated vesicles, respectively.
在0℃下，使用抗毒素125I-蛋白A测定法追踪与神经元细胞结合的破伤风毒素的命运。将细胞升温至37℃后5分钟内，约50%的表面结合毒素消失。用125I-毒素进行的实验表明，这种损失大部分是由于结合毒素解离到培养基中。然而，一些毒素迅速被内化，只有在用Triton X-100使细胞通透后进行测定才能检测到。为了研究内化机制，将破伤风毒素吸附到胶体金上。毒素-金显示是稳定的，并且与游离毒素识别相同的受体。在4℃下对结合到细胞体上的毒素-金颗粒分布进行定量分析表明，它集中在被膜小窝中。在37℃下5分钟后，毒素-金出现在被膜小泡、内体和小管中。15分钟后，它主要存在于内体中，30分钟后存在于多泡体中。使用游离毒素、抗毒素和蛋白A-金证实了被膜小窝参与破伤风毒素而非霍乱毒素的内化。毒素-金也通过被膜小窝进入神经末梢和轴突，分别积聚在突触小泡和轴突内无被膜小泡中。
