title: Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors.
abstract: 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.
  
  
  
  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).
  
  
  
  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 Kmvalue of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The Kmvalue of the mutant (Serine to Glycine) increased to 0.19 mM. The Kmvalue (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.
  
  
  
  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: Department of Parasitology, Faculty of Medicine, University of Peradeniya, Peradeniya 20400, Sri Lanka. Electronic address: susijij@pdn.ac.lk.
authors: Susiji
authors: S
authors: Wickramasinghe
authors: Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, University of Peradeniya, Peradeniya 20400, Sri Lanka.
authors: Lalani
authors: L
authors: Yatawara
authors: Department of Environmental Health Sciences, Kochi Medical School, Oko, Nankoku City, Kochi Ken 783-8505, Japan.
authors: Mitsuru
authors: M
authors: Nagataki
authors: Department of Environmental Health Sciences, Kochi Medical School, Oko, Nankoku City, Kochi Ken 783-8505, Japan.
authors: Takeshi
authors: T
authors: Agatsuma
keywords: Arginine kinase; Gene structure; Inhibition kinetics; Site directed mutagenesis; Toxocara canis