J S Knight

Publications (3)6.37 Total impact

• Article: Molecular and biochemical characterisation of arginine kinases in Haemonchus contortus and Teladorsagia circumcincta.

  S Umair, J S Knight, R J Bland, H V Simpson
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  ABSTRACT: Full length cDNA encoding arginine kinases (AK) were cloned from Teladorsagia circumcincta (TcAK) and Haemonchus contortus (HcAK). The TcAK and HcAK cDNA (1080bp) encoded 360 amino acid proteins. The predicted amino acid sequence showed 99% similarity with each other and 94% with a Caenorhabditis elegans AK. Soluble N-terminal His-tagged AK proteins were expressed in Escherichia coli strain BL21, purified and characterised. All binding sites were completely conserved in both proteins. The recombinant TcAK and HcAK had very similar kinetic properties: Km arginine was 0.35mM, Km ATP was 0.8-0.9mM and the pH optima were pH 7.5. Arginine analogues strongly inhibited recombinant enzyme activities (up to 80%), whilst other amino acids decreased activities by a maximum of 20%. TcAK and HcAK are potential vaccine candidates because of the strong antigenicity of invertebrate phosphagens and kinases and presence in metabolically active parts of worm.
  Experimental Parasitology 04/2013; · 2.12 Impact Factor
• Article: Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta.

  S Umair, C Ria, J S Knight, R J Bland, H V Simpson
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  ABSTRACT: Sarcosine (N-methylglycine) is an intermediate in glycine degradation and can also be synthesised from glycine in mammals. Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta differed from that of mammals in that creatinase activity was present and sarcosine was demethylated only by sarcosine oxidase (SOX) and not by sarcosine dehydrogenase (SDH). The mean SOX activity was 30 nmoles min(-1) mg(-1) protein in homogenates of L3 and adult worms of both parasites and the apparent K(m) for sarcosine was 1.1 mM. Addition of 2 mM Cd(2+) inhibited activity by 30%. There was no SDH activity with either NAD(+) or NADP(+) as co-factor. Mean creatinase activity in L3 T. circumcincta and adult worms of both species was 31 ± 6 nmoles min(-1) mg(-1) protein, but was undetectable in L3 H. contortus. Activity was inhibited by up to 70% by Cu(2+), Fe(2+), Fe(3+) and Zn(2+). Possessing creatinase would allow host creatine to be incorporated into amino acids by the parasites.
  Experimental Parasitology 01/2013; · 2.12 Impact Factor
• Article: Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase.

  S Umair, J S Knight, M L Patchett, R J Bland, H V Simpson
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  ABSTRACT: A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD(+) and NADP(+), GDH activity was greater in the deaminating reaction with NADP(+) as co-factor and more with NADH in the aminating reaction.
  Experimental Parasitology 08/2011; 129(3):240-6. · 2.12 Impact Factor