Evans D M, Williams R S, Shone C C, Hambleton P, Melling J, Dolly J O
Eur J Biochem. 1986 Jan 15;154(2):409-16. doi: 10.1111/j.1432-1033.1986.tb09413.x.
Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.
通过亲和色谱和离子交换色谱法将来自B型肉毒梭菌的神经毒素纯化至同质；该蛋白（Mr约等于155000）经胰蛋白酶消化后的比神经毒性达到2×10⁸小鼠LD₅₀单位/毫克蛋白的水平。将毒素进行¹²⁵I碘化以获得高比放射性（19 - 63 TBq/mmol），其毒性通常保留大于65%；在还原条件下经胰蛋白酶消化后进行十二烷基硫酸钠凝胶电泳，结果显示较大的多肽（Mr约等于101000）被优先标记。观察到¹²⁵I标记的神经毒素与大鼠大脑皮质突触体存在可饱和结合，Scatchard分析表明受体含量低但亲和力高（Kd = 0.3 - 0.5 nM；Bmax约等于30 - 60 fmol/毫克蛋白），同时还有大量低亲和力位点。在使用天然或完全活化（经胰蛋白酶处理）的神经毒素进行竞争实验时，未观察到结合亲和力有显著差异，这表明链裂解对于受体 - 毒素相互作用并非必需。A型肉毒杆菌神经毒素即使在高浓度（1 μM）时抑制标记的B型毒素与突触体结合的能力也有限，而其他神经毒素则无作用，这突出了受体的选择性。用神经氨酸酶预孵育突触体可导致特异性毒素结合几乎完全丧失，而用麦胚凝集素抑制低亲和力位点的作用则不太明显；分别加入选择性抑制剂（2,3 - 脱氢 - 2 - 脱氧 - N - 乙酰神经氨酸和N - 乙酰葡糖胺）可防止这种失活。这些观察结果表明N - 乙酰神经氨酸以及可能的其他糖部分是毒素受体的组成成分。当用1 nM或10 nM¹²⁵I碘化毒素检测时，突触体经胰蛋白酶消化后对结合的抑制不完全。对神经氨酸酶、胰蛋白酶和热处理对毒素结合浓度依赖性的作用进行详细分析表明，至少存在两个可区分的含N - 乙酰神经氨酸的位点群体，其中一个蛋白质成分与较低亲和力的受体相关。结合先前关于A型神经毒素的报道以及此类膜受体可能的生理意义对这些发现进行了讨论。
