Hussein A S, Grigg M E, Selkirk M E
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Exp Parasitol. 1999 Feb;91(2):144-50. doi: 10.1006/expr.1998.4360.
We have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic (G1na) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts of N. brasiliensis and report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (G4a) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 microM for acetylthiocholine was determined. It was highly sensitive to the AChE-specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 microM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50 of 46.5 +/- 6.1 microM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.
我们之前已确定，巴西日圆线虫分泌三种单体非两亲性（G1na）变体的乙酰胆碱酯酶（AChE），其性质大致相似。在本研究中，我们检测了巴西日圆线虫体细胞提取物中的AChE表达，并报告鉴定出一种未分泌的额外酶。该酶通过蔗糖密度梯度离心分离，沉降系数为10.2 S，在Triton X - 100存在下转移至9.4 S，确定该酶为四聚体两亲性（G4a）形式。通过电荷转移电泳证实了该酶的两亲性，其中与脱氧胆酸钠相互作用加速了迁移。该酶对丁酰硫代胆碱活性较低，测定其对乙酰硫代胆碱的米氏常数为91±13μM。它对AChE特异性抑制剂双（4 - 烯丙基二甲基铵苯基）戊 - 3 - 酮二溴化物高度敏感，IC50为6.5±0.4μM，但也被丁酰胆碱酯酶特异性抑制剂四单异丙基焦磷酰胺抑制，尽管IC50较高，为46.5±6.1μM。因此，这种酶可通过其两亲性、在蔗糖梯度中的沉降以及对胆碱酯酶抑制剂的敏感性与分泌的AChEs区分开来。
