Rhoads M L
J Biol Chem. 1981 Sep 10;256(17):9316-23.
An antigenic secretory protein with cholinesterase activity was isolated from the excretory gland cells of Stephanurus dentatus and was purified by gel filtration and ion exchange chromatography. The antigenicity of the cholinesterase was demonstrated by an esterase-active immunoprecipitate formed with S. dentatus antiserum and by the ability of the antiserum to protect the enzyme from heat inactivation. The enzyme was found to be secreted by the adult nematodes during in vitro cultivation. The level of cholinesterase activity and its release from the excretory gland cells of the parasite were 27-fold greater in the male than in the female. Ninety per cent of the enzyme activity was localized in the soluble fraction of the gland cells. The molecular weight of the enzyme, estimated by sucrose density gradient centrifugation, was 100,000. Two molecular forms were separated by isoelectrofocusing, with isoelectric points of 7.0 and 6.9. At optimum substrate concentrations, the rate of hydrolysis of acetylthiocholine was 8 times greater than that of butyrylthiocholine; the Michaelis constants were 560 microM and 81 microM for acetylthiocholine and butyrylthiocholine, respectively. The enzyme exhibited substrate inhibition at substrate concentrations greater than 10 mM and was inhibited by eserine sulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, Tris, and acetone. The enzyme was highly unstable in dilute protein solutions.
从有齿冠尾线虫的排泄腺细胞中分离出一种具有胆碱酯酶活性的抗原性分泌蛋白，并通过凝胶过滤和离子交换色谱法进行纯化。胆碱酯酶的抗原性通过与有齿冠尾线虫抗血清形成的酯酶活性免疫沉淀物以及抗血清保护该酶免受热失活的能力得以证明。发现该酶是成年线虫在体外培养期间分泌的。寄生虫排泄腺细胞中胆碱酯酶活性水平及其释放量在雄性中比雌性高27倍。90%的酶活性定位于腺细胞的可溶性部分。通过蔗糖密度梯度离心法估计，该酶的分子量为100,000。通过等电聚焦分离出两种分子形式，其等电点分别为7.0和6.9。在最佳底物浓度下，乙酰硫代胆碱的水解速率比丁酰硫代胆碱高8倍；乙酰硫代胆碱和丁酰硫代胆碱的米氏常数分别为560 microM和81 microM。该酶在底物浓度大于10 mM时表现出底物抑制作用，并受到硫酸依色林、1,5-双(4-烯丙基二甲基铵苯基)-戊烷-3-酮二溴化物、Tris和丙酮的抑制。该酶在稀释的蛋白质溶液中高度不稳定。
