Arginine kinase in Toxocara canis: Exon–intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors
10.1016/j.apjtm.2016.07.023
- Author:
Susiji WICKRAMASINGHE
1
;
Lalani YATAWARA
2
;
Mitsuru NAGATAKI
3
;
Takeshi AGATSUMA
3
Author Information
1. Department of Parasitology, Faculty of Medicine, University of Peradeniya
2. Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, University of Peradeniya
3. Department of Environmental Health Sciences, Kochi Medical School
- Publication Type:Journal Article
- Keywords:
Arginine kinase;
Gene structure;
Inhibition kinetics;
Site directed mutagenesis;
Toxocara canis
- From:
Asian Pacific Journal of Tropical Medicine
2016;9(10):995-1001
- CountryChina
- Language:Chinese
-
Abstract:
Objectives 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 BamH1/SalI site of pMAL-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 pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C 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
