Pa-11在神經鍵體細胞膜上之專一性結合

Abstract

Pa-11是由澳洲大棕蛇(Pseudechis australis)純化出來的鍵前神經蛇毒，碘化後的 Pa-11 在天竺鼠神經鍵體細胞膜上具有親和力極高的專一性結合。此專一性結合由 scatchard plots 圖形上，可以解析為二種獨立的結合位置。以 Rosenthal 氏圖形分析法求得 125I-Pa-11 在這二位置的解離常數分別為：Kd1= 0.25 ± 0.03 nM 及Kd2= 4.6 ± 0.8 nM；最大結合量分別為：Bmaxl= 6.9 ± 0.04 pmole/mg of protein 及 Bmax2=23.4± 2.1 pmole/mg of protein。 加熱處理神經鍵體細胞膜，可以顯著降低 125I-Pa-11 的結合量，但以 protease 處理時則否。125I-Pa-11 在神經鍵體細胞膜的專一性結合，隨著一價陽離子的濃度而改變；約在 100 mM時，專一性結合的量達最高；此結合並不完全依賴於鈣離子，因為在 10 mM EDTA 存在下，仍有專一性結合；此外，在結合反應中除了鎂離子外，鍶離子及鋇離子均可取代鈣離子。 未標示的 Pa-11 和大部分測試的 PLA2 均可抑制 125I-Pa-11 在神經鍵體細胞膜的專一性結合，惟其抑制的能力不一。以串接試劑親和性標示125I-Pa-11在神經鍵體細胞膜上的結合體，可以得到 73 kDa 及 34 kDa 的二個聯結體，但是加熱或以 protease 處理神經鍵體細胞膜後即無法生成；其他組織的細胞膜也可以生成這二個聯結體。同樣地，能抑制125I-Pa-11 結合的 PLA2也能抑制這二個聯結體的生成，而且它們抑制兩者的能力相近。

Pa-l1, a presynaptic neurotoxin from Pseudechis australis, was iodinated and used to demonstrate high affinity, specific binding to synaptic membrane. The binding curve in Scatchard plots can be resolved into two binding site. The dissociation constants are Kd1 = 0.25 ± 0.03 nM and Kd2= 4.6 ± 0.8 nM, and the maximal binding capacities are Bmax1 = 6.9 ± 0.4 and Bmax2= 23.4 ± 2.1 pmol/ mg of protein by Rosenthal analyses . The binding of 125I-Pa-11 to synaptic membranes is sensitive to heat but resistant to proteolysis. Monovalent cations influence the 125I-Pa-11 binding in a concentration dependent manner with optimal binding ocurring at 100 mM. Sr2+ and Ba2+ but not Mg2+ can replace Ca2+ in the binding reaction, but the binding does not disappear even in the absence of divalent cations.Competition experiments have shown that almost all the PLA2 tested can displace the 125I-Pa-11 binding, though with different capacities. Cross-linking of the toxin to its acceptors in the synaptic membrane, reveals two major adducts of 73 and 34 kDa,which are susceptible to heat and protease, and are observed with other tissuew as well. Other PLA2 can displace these adducts formation with the same order of potency as in compectitive binding experiments.