Vernon L P, Rogers A
Chemistry Department, Brigham Young University, Provo, UT 84602.
Toxicon. 1992 Jul;30(7):701-9. doi: 10.1016/0041-0101(92)90004-o.
Pyrularia thionin is a strongly basic bioactive peptide of 47 amino acids isolated from nuts of Pyrularia pubera. It is hemolytic, cytotoxic and activates an endogenous phospholipase A2 in 3T3 cells. Earlier studies have shown that the cardiotoxin from Naja naja kaouthia has similar activities and binds to the same site as Pyrularia thionin. Since the peptides appear to bind to the phospholipids of cell membranes to elicit their cellular responses, the effect of modifying the electrostatic environment was studied by separately adding phosphate ion and Ca2+, and by removing Ca2+ from the membrane by treatment with EGTA. Analysis of erythrocyte hemolysis for both Pyrularia thionin and cardiotoxin shows that the reactions follow Michaelis-Menten kinetics, with the peptides serving as the substrate. The basal rate of hemolysis in physiological saline is markedly increased by the addition of phosphate in the 5-10 mM range and also by removing membrane-bound Ca2+ by incubation of the cells with 10 mM EGTA. These treatments do not change the apparent K(m) values, but increase the V(max), indicating that more binding sites are made available by these treatments. On the other hand, added Ca2+ in the 5-10 mM range competitively inhibits the reaction by inhibiting the binding of the peptide to the membrane.
山桐子硫堇是一种从毛叶山桐子坚果中分离得到的由47个氨基酸组成的强碱性生物活性肽。它具有溶血、细胞毒性，并能激活3T3细胞中的内源性磷脂酶A2。早期研究表明，眼镜蛇印度亚种的心脏毒素具有类似的活性，且与山桐子硫堇结合于同一部位。由于这些肽似乎通过结合细胞膜磷脂来引发细胞反应，因此通过分别添加磷酸根离子和Ca2+，以及用EGTA处理细胞膜以去除Ca2+来研究改变静电环境的影响。对山桐子硫堇和心脏毒素的红细胞溶血分析表明，反应遵循米氏动力学，这些肽作为底物。在生理盐水中，添加5 - 10 mM范围内的磷酸盐以及用10 mM EGTA孵育细胞以去除膜结合的Ca2+，均可显著提高基础溶血速率。这些处理不会改变表观K(m)值，但会增加V(max)，表明这些处理可提供更多的结合位点。另一方面，添加5 - 10 mM范围内的Ca2+会通过抑制肽与膜的结合而竞争性抑制反应。
