雨傘節突觸前神經毒族群中神經毒性的研究

Abstract

雨傘節毒液中，存在一種β-BuTX的毒蛋白，具有PLA2的活性，屬於突觸前神經毒，其分子由兩個subunit以雙硫鍵連結而成，一為具有120個胺基酸的A-subunit，結構類似於bovine pancreatic PLA2；另一為具有60個胺基酸的B-subunit，結構類似於kunitz type trypsin inhibitor familiy。 我們利用Sephdex G-50 column將毒液含20 KDa蛋白分離，再將此蛋白部分通過SP column以高效能液相層析法（HPLC）分離，於層析圖上出現至少十八個波峰，依順序定為P1-P18,P3-P18經鑑定為β-BuTX isotoxin到至少十六種的β-BuTX isotoxin ，對部分的毒蛋白做LD50毒性，PLA2酵素活性及小雞神經肌肉收縮的抑制等測試，發現毒性最弦的毒蛋白P16，毒性強度遠比過去曾經報告過的β-BuTx的毒性為毒，幾乎是毒性最弱的毒蛋白P4的1000 倍。且毒蛋白的毒性會隨著毒蛋白的鹼性而有增強的趨勢。 毒蛋白P4-P11在PLA2酵素活性及小雞神經肌肉的實驗中，酵素活性和毒性隨著毒蛋白鹼性的增加而有增加的趨努，和LD550實驗的結果有一致性，但P13-P18在PLA2 酵素活性及小雞神經肌肉的實驗結果，卻無上述的趨勢。毒蛋白P4、P5在LD50的實驗中，老鼠經注射後毒發死亡的時間遠比P8、P11及其他毒蛋白為長。為了比較毒蛋白P4和P8之間在突觸膜上毒性競爭作用的能力，我們利用小雞神經肌肉收縮的實驗，發現P8的作用能力遠大於P4，由於 P4及P8是由不同的A和相同的B subunit所組成，他們在突觸膜上是否作用於同一個或是不同的acceptor，則有待進一步探討。此外，用同樣的條件比較毒白P5及P11作用至突觸膜的能力，發現P11的作用能力遠大於P5。目前分離出的β-BuTX，至少存在十六種以上的isoform，若是進一步研究其胺基酸組成，應可發現新種類的A及B subunit，若配合本篇論文的結果，應有助於在分子層次上對β-BuTX的瞭解。

These exists in the venom of bandit krait (Bungarus multicinctus), a group of polypeptide toxins, which act presynaptically to block the transmission between the neuromuscular junction. These toxins are generally named as the isotoxins of β-bungarotoxin family, The toxin molecule composes of two subunits which are linked together by a disulfide bond. One is A chain which has 120 amino acids with high similarity to the primary structure of vertebrate PLA2 enzyme. The other is B chain, which has 60 amino acids with high similarity to the primary kunitz-type trypsin inhibitor. By Sephadex G-50 column chromatography and HPLC on a SP column, we were able to identify at least sixteen isotoxins of β-BuTX family in one bench of venom available from commercial source (Chen,chung chu, 94). We assayed their functional characteristics including PLA2 enzyme activity, lethal potency and the inhibitory effect on the indirectly evoked contraction of chicken biventer muscle. We found that the most toxic protein, P16, had LD50 far less than that reported for β-BuTX which was 1000 folds more toxic than the weakest P4. Approximately, the lethality increased in accordance with the basicity of isotoxin. P4 and P5 showed weaker PLA2 enzyme activity than that of P11 which was demostrated to be β-BuTX reported in the literature. P4 and P5 retained 17.7% and 34.3% of PLA2 activity of P11. P13-P18 all had comparable enzyme activity. In the LD50 experiment, the mouse injected with P4, P5 died much later than those injected with P8, P11 and other isotoxins. The ability of each isotoxin to block the transmission between nerve and muscle was generally parallel to its LD50.