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

Publication Overview
TitleArginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors
AuthorsWickramasinghe S, Yatawara L, Nagataki M, Agatsuma T
TypeJournal Article
Journal NameAsian Pacific journal of tropical medicine
Volume9
Issue10
Year2016
Page(s)995-1001
CitationWickramasinghe S, Yatawara L, Nagataki M, Agatsuma T. Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors. Asian Pacific journal of tropical medicine. 2016 Oct; 9(10):995-1001.

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 (1200 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 Km value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The Km 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-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T. canis.

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This publication contains information about 1 features:
Feature NameUniquenameType
Tf0151Tf0151genetic_marker
Properties
Additional details for this publication include:
Property NameValue
Publication ModelPrint-Electronic
ISSN2352-4146
eISSN2352-4146
Publication Date2016 Oct
Journal AbbreviationAsian Pac J Trop Med
PIIS1995-7645(16)30161-4
Elocation10.1016/j.apjtm.2016.07.023
DOI10.1016/j.apjtm.2016.07.023
Publication TypeJournal Article
Journal CountryChina
CopyrightCopyright © 2016 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.
LanguageEnglish
Language AbbrENG
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PMID: PubMedPMID:27794395