Simpson L L, Lautenslager G T, Kaiser I I, Middlebrook J L
Department of Medicine, Jefferson Medical College, Philadelphia, PA 19107.
Toxicon. 1993 Jan;31(1):13-26. doi: 10.1016/0041-0101(93)90352-j.
Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.
使用五种具有不同链结构和抗原性的磷脂酶A2神经毒素[诺维毒素（单链）；响尾蛇毒素（两条非共价结合的链），β-银环蛇毒素（两条共价结合的链）；太攀蛇毒素（三条链），以及织绵蛇毒素（五条链；三条链各一份，第四条链两份）]对小鼠半膈肌标本进行了实验。在对比实验中使用了三种梭菌神经毒素（A型和B型肉毒杆菌神经毒素以及破伤风毒素）。磷脂酶A2神经毒素产生浓度依赖性的神经肌肉传递阻滞。链结构与效力之间没有明显关系，但有迹象表明链结构与结合之间存在关系。诺维毒素的结合基本上是可逆的，响尾蛇毒素的结合略有可逆性，而β-银环蛇毒素、太攀蛇毒素和织绵蛇毒素的结合则很难逆转。用中和抗体进行的实验表明，磷脂酶A2神经毒素与细胞表面或其附近的结合位点相关联。这种结合并未产生神经肌肉阻滞。当暴露于生理温度和神经刺激时，结合的毒素从可被中和抗体识别的状态消失。这一发现表明存在某种形式的分子重排。两种最有可能的可能性是：（1）毒素分子的构象发生了变化，或者（2）毒素与膜之间的关系发生了变化。分子重排步骤并未产生神经肌肉阻滞。在稍后的时间出现了麻痹；阻滞开始所需的时间是毒素浓度的函数。磷脂酶A2神经毒素不受抑制受体介导的内吞作用的药物的拮抗。此外，磷脂酶A2神经毒素并未表现出其他内吞蛋白质（如梭菌神经毒素）典型的pH诱导的构象变化。然而，磷脂酶A2神经毒素受到锶的拮抗，这种拮抗作用针对溶液中的游离毒素和结合在细胞表面的毒素。在毒素经历分子重排后表现出有限的拮抗作用，而在毒素诱导神经肌肉阻滞后则没有拮抗作用。累积的数据表明，磷脂酶A2神经毒素不会被内化以产生其中毒作用。这些毒素似乎作用于质膜，这是它们引发最终导致神经肌肉阻滞的事件的部位。
