Islam M Khyrul, Miyoshi Takeharu, Kasuga-Aoki Harue, Isobe Takashi, Arakawa Takeshi, Matsumoto Yasunobu, Tsuji Naotoshi
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5, Kannondai, Tsukuba, Ibaraki, Japan.
Eur J Biochem. 2003 Jul;270(13):2814-26. doi: 10.1046/j.1432-1033.2003.03658.x.
Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli-expressed recombinant enzyme has a specific activity of 937 micro mol Pi.min-1.mg-1 protein corresponding to a kcat value of 638 s-1 at 55 degrees C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.
无机焦磷酸酶（PPase）是一种重要的酶，可催化无机焦磷酸（PPi）水解为正磷酸盐（Pi）。我们在此报告了编码猪蛔虫可溶性PPase的基因的分子克隆及特性分析。预测的猪蛔虫PPase由360个氨基酸组成，分子量为40.6 kDa，pI为7.1。氨基酸序列比对和系统发育分析表明，该基因编码一种功能性的I型可溶性PPase，其特征与原核、植物及动物/真菌可溶性PPase相同。在大肠杆菌中表达的重组酶在55℃时的比活性为937微摩尔Pi·分钟-1·毫克-1蛋白质，对应kcat值为638秒-1。其活性强烈依赖于Mg2+，并受到Ca2+的抑制。天然PPase在猪蛔虫的所有发育阶段均有表达。在与之关系最密切的人类和犬类蛔虫——人蛔虫和犬弓首蛔虫中也分别检测到了一个同源物。该酶在成年雌虫的体壁、肠上皮、卵巢和子宫中高度定位。我们观察到，亚氨基二磷酸和氟化钠（可溶性和膜结合H+-PPase的有效抑制剂）以剂量依赖方式有效抑制了猪蛔虫L3至L4体外的天然PPase活性以及发育和蜕皮。这些研究提供了证据，表明PPase是蛔虫中的新型酶，可能在发育和蜕皮过程中起关键作用。
