澳州紅腹黑蛇神經毒素在神經鍵體上之專一性結合

Abstract

Pseudexin B是由澳洲紅腹黑蛇（Pseudechis porphyriacus）純化出來的神經毒素，碘化後的pseudexin B在天竺鼠大腦的神經鍵體上有專一性結合。此專一性結合在Scatchard plot上可分解成兩種不同之親和力，以Rosenthal圖形法可求得這兩個解離常數分別?：Kd1=1.2±0.05nM及Kd2=10±3nM ；最大結合量分別為：Bmax1=2±0.15pmol/mg of protein及Bmax2=77±3pmol/mg of protein。加熱或以proteases處理神經鍵體後125I-pseudexin B在神經鍵體上的結合量沒有明顯下降。此專一性結合會隨著一價正離子濃度的改變而升降，約在100mM的結合量最大。二價正離子中，鍶離子及鋇離子約可取代鈣離子，而鎂離子及鈷離子則否。 許多其他PLA2分子均可抑制125I-pseudexin B在神經鍵體上的結合，但β-bungarotoxin及 notexin則否。以化學串接試劑進行共價聯結實驗標示125I-pseudexin B在神經鍵體上的接受體，可看到一條分子量為40KDa的共價聯結體。以proteases處理後，此標示帶即消失。在結合實驗中可抑制125I-pseudexin B結合的PLA2分子，也可抑制此一標示帶的生成，且其抑制能力之強弱與結合實驗求得之趨勢一致。

Pseudexin B was purified from the venom of Pseudechis porphriacus, and then iodinated for study of its binding to membranes from the brain . Specific binding of 125I-pseudexin B to guinea pig synaptosomes was demonstrated , and the binding curve could be resolved into two families of binding sites with Kd1=1.2±0.05nM and Kd2=10±3nM .The maximal binding Capacities were Bmax1=2±0.15 and Bmax2=77±3pmol/mg of protein. The amount of 125I-pseudexin B bound to synaptosomes was dependent on monovalent cation concentrations and with optimal concentration about 100mM Na+. Sr2+ and Ba2+ could, while Mg2 + and Co2 + could not replace Ca2 + in the binding reaction. The synaptosome binding sites were not sensitive to proteases and heat. One major conjugate of 40KDa was obtained when 125I-pseudexinB was cross-linked to its binding sites on membranes of synaptosomes . One major conjugate of 40KDa was obtained. As in binding experiments, this conjugation was prevented by most snake venom PLA2, but β-bungarotoxin and notexin were not inhibitory.