Arginine kinase in Toxocara canis:Exon-intron organization,functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors

作     者：Susiji Wickramasinghe Lalani Yatawara Mitsuru Nagataki Takeshi Agatsuma 

作者机构：Department of ParasitologyFaculty of MedicineUniversity of Peradeniya Department of Medical Laboratory sciencesFaculty of Allied Health SciencesUniversity of Peradeniya Department of Environmental Health SciencesKochi Medical School 

出 版 物：《Asian Pacific Journal of Tropical Medicine》 (亚太热带医药杂志（英文版）)

年 卷 期：2016年第9卷第10期

页      面：973-979页

核心收录：

学科分类：1001[医学-基础医学(可授医学、理学学位)] 100103[医学-病原生物学] 10[医学] 

基　　金：Japan Society for the Promotion of Science  JSPS  (26305011) 

主　　题：Toxocara canis Arginine kinase Gene structure Site directed mutagenesis Inhibition kinetics 

摘      要：Objective: To determine exon/intron organization of the Toxocara canis(T. canis) AK(TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Methods: Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame(1 200 bp) of TCAK(wild type) was cloned into the BamH 1/SalI site of pM AL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pM AL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25℃ for the forward reaction(phosphagen synthesis). Results: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K_m value of the mutant(Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K_m value of the mutant(Serine to Glycine) increased to 0.19 mM. The Km value(0.19 mM) of the double mutant(Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type(0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica(A. indica), an aminoglycoside antibiotic(aminosidine), a citrus flavonoid glycoside(rutin) and a commercially available catechin mixture against TCAK. Green and black tea(1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Conclusions: Arginine kinase in T. canis has a seven-exon/six