Arginine kinase in Toxocara canis: Exon–intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors
Journal Title: Asian Pacific Journal of Tropical Medicine - Year 2016, Vol 9, Issue 10
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 noncompetitive 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.
Authors and Affiliations
Susiji Wickramasinghe
Keywords
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- EP ID EP282587
- DOI 10.1016/j.apjtm.2016.07.023
- Views 71
- Downloads 0
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