Identification of GR and TrxR systems in Setaria cervi : Purification and characterization of glutathione reductase

• Published in: Parasitology international Vol. 62; no. 2; pp. 193 - 198
• Main Authors: Yadav, Sudhanshu S, Srikanth, Elesela, Singh, Neetu, Rathaur, Sushma
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ireland Ltd 01.04.2013
• Subjects: activation energy; active sites; adults; ammonium sulfate; Animals; antioxidants; Catalytic Domain; Cattle; Chromatography, Affinity; Chromatography, Ion Exchange; cysteine; Drug target; drugs; enzyme activity; Enzyme Inhibitors - pharmacology; Enzyme kinetics; Female; Filariasis; Filariasis - parasitology; fractionation; Gastroenterology and Hepatology; glutathione; Glutathione reductase; Glutathione Reductase - isolation & purification; Glutathione Reductase - metabolism; glutathione-disulfide reductase; histidine; Hydrogen-Ion Concentration; Infectious Disease; inhibitory concentration 50; ion exchange chromatography; Kinetics; Life Cycle Stages; Male; microfilariae; Molecular Weight; NADP; NADP (coenzyme); Oxidation-Reduction; parasites; parasitology; polyacrylamide gel electrophoresis; Setaria; Setaria cervi; Setaria Nematode - enzymology; Setaria Nematode - growth & development; Temperature; Thioredoxin reductase; Thioredoxin-Disulfide Reductase - isolation & purification; Thioredoxin-Disulfide Reductase - metabolism; tyrosine; Drug target; Filariasis; Enzyme kinetics; Glutathione reductase; Thioredoxin reductase
• ISSN: 1383-5769; 1873-0329
• DOI: 10.1016/j.parint.2012.12.008

Abstract The glutathione reductase (GR) and thioredoxin reductase (TrxR) are important enzymes of the redox system that aid parasites to maintain an adequate intracellular redox environment. In the present study, the enzyme activity of GR and TrxR was investigated in Setaria cervi ( S. cervi ). Significant activity of both enzymes was detected in the somatic extract of adult and microfilariae stages of S. cervi . Both GR and TrxR were separated by partial purification using ammonium sulfate fractionation and DEAE ion exchange chromatography suggesting the presence of both glutathione and thioredoxin systems in S. cervi . The enzyme glutathione reductase (ScGR) was purified to homogeneity using affinity and ion exchange chromatography that resulted in 90 fold purification with a yield of 11.54%. The specific activity of the ScGR was 643 U/mg that migrated as a single band on SDS-PAGE. The subunit molecular mass was determined to be ~ 50 kDa while the optimum pH and temperature were found to be 7.0 and 35 °C respectively. The activation energy (Ea) was calculated from the slope of Arrhenius plot as 16.29 ± 1.40 kcal/mol. The Km and Vmax were determined to be 0.27 ± 0.045 mM; 30.30 ± 1.30 U/ml with NADPH and 0.59 ± 0.060 mM; 4.16 ± 0.095 U/ml with GSSG respectively. DHBA, a specific inhibitor for GR has completely inhibited the enzyme activity at 1 μM concentration. The inhibition of ScGR activity with NAI (IC50 0.71 mM), NEM (IC50 0.50 mM) and DEPC (IC50 0.27 mM) suggested the presence of tyrosine, cysteine and histidine residues at its active site. Further studies on characterization and understanding of these antioxidant enzymes may lead to designing of an effective drug against lymphatic filariasis.