Analysis of physiological functions of target proteins of tetanus and diphtheria toxins using complexes of homologous fragments of the toxins

用二硫苏糖醇（DTT）还原和4 M-2 M尿素处理破伤风和白喉毒素，经Mono Q柱分离，得到两个互补的多肽片段。通过使用CDI [1-乙基-3-（3-L-二甲基氨基丙基）马来酰亚胺-HCl]或SPDP [N-琥珀酰亚胺基3-（2-吡啶基二硫代）丙酸酯]或通过氧化和通过透析从解离的毒素的混合物中除去尿素来制备由来自不同毒素的N-末端酶活性框架和C-末端细胞结合片段组成的复合物。结果表明，由不同来源的酶活性片段和细胞结合片段组成的复合物对Vero细胞和PC_（12）细胞的毒性、对豚鼠的皮肤坏死活性和致死性、神经毒性均较低。但用CDI制备的白喉毒素片段A或白喉毒素与神经生长因子的复合物引起胆碱能神经元特异性的细胞毒性，表明用CDI制备的复合物中的片段A保留了其酶活性。来自DTT还原的尿素解离的毒素的混合物的重构复合物的混合物似乎显示出对Vero细胞的细胞毒性增加。因此，目前正在研究重组和重组材料的分离和纯化制剂的细胞毒性。纯化的重组毒素对神经分泌型PC 12细胞释放去甲肾上腺素的影响，以及这些物质对豚鼠和白喉毒素抗性大鼠和小鼠的致死性神经毒性活性，正在与针对每个毒素片段的抗体的中和作用一起进行研究。

Tetanus and diphtheria toxins were highly purified by procedures includind HPLC.Two complementary polypeptide fragments from each toxin were separated and highly purified, using a Mono Q column in an FPLC system, from toxins dissociated by reduction with dithiothreitol (DTT) and treatment with 4M-2M urea. Complexes composed of an N-terminal enzymatic, active frament and a C-terminal cell-binding fragment from the different toxins were prepared by using CDI [1-ethyl-3- (3-L-dimethyl aminopropyl) carbodiimide-HCl] or SPDP [N-succinimidyl 3- (2-pyridyldithio) propionate] or by oxidation and removing urea by dialysis from a mixture of the dissociated toxins. Cytotoxicity on Vero and PC12 cells, dermonecrotizing activity and lethal, neurotoxic activities on guinea pigs of the complexes were examined, In contrast to the oridinal toxins and the respectivereconstituted toxins, these complexes composed of an enzymatic active fragment and a cell-binding fragment from different origins elicited only slight toxicity. But the complexes composed of the diphtheria toxin fragment A or diphrheria toxin and a nerve growth factor prepared by using CDI elicited cholinergic neuron-specfic cytotoxicity, indicating that the fragment A in the complex pepeared using CDI retained its enzymatic activity. The mixture of reconstituted complexes from the mixture of DTT-reduced, urea-dissociated toxins appeared to show an increase in cytotoxicity on Vero cells. So the cytotoxicity of the isolated and purified preparations of reconstituted and recombined materials are now being investigaed. The effects of the purified recombined toxins on noradrenaline release from neurosecretoy PC12 cell and lethal, neurotoxic activity of these materials on guinea pigs and diphtheria toxin-resistant rats and mice are being investigated together with the neutralizing effects of antibodies against each toxin-fragment.