The glutathione S-transferase from Plasmodium falciparum

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dc.contributor.author Liebau, Eva
dc.contributor.author Campbell, Alison M.
dc.contributor.author Bergmann, B.
dc.contributor.author Luersen, K.
dc.contributor.author Brophy, Peter M.
dc.contributor.author Teesdale-Spittle, Paul
dc.contributor.author Walter, R. D.
dc.date.accessioned 2010-02-05T15:38:27Z
dc.date.available 2010-02-05T15:38:27Z
dc.date.issued 2002
dc.identifier.citation Liebau , E , Campbell , A M , Bergmann , B , Luersen , K , Brophy , P M , Teesdale-Spittle , P & Walter , R D 2002 , ' The glutathione S-transferase from Plasmodium falciparum ' Molecular and Biochemical Parasitology , pp. 1-2 . , 10.1016/S0166-6851(02)00160-3 en
dc.identifier.other PURE: 134793
dc.identifier.other dspace: 2160/4043
dc.identifier.uri http://hdl.handle.net/2160/4043
dc.description Liebau, E., Bergmann, B., Campbell, A. M., Teesdale-Spittle, P., Brophy, P. M., Luersen, K., Walter, R. D. (2002). The glutathione S-transferase from Plasmodium falciparum. Molecular and Biochemical Parasitology, 124, (1-2), 85-90 en
dc.description.abstract Malaria is one of the most important widespread diseases in the world, with about 300–500 million clinical cases and 1.7–2.5 million deaths every year. Plasmodium falciparum is the causative agent of the most severe form of malaria. The rapidly developing resistance to drugs used for prophylaxis and treatment makes the identification of novel drug targets necessary [1]. The glutathione S-transferases (GSTs) [EC 2.5.1.18] represent a large family of enzymes found in organisms ranging from prokaryotes to mammals. They detoxify both endogenous and exogenous xenobiotic compounds via the nucleophilic addition of GSH to a large variety of electrophilic substrates. Besides catalyzing conjugation reactions, GSTs possess selenium-independent glutathione peroxidase (GPx) activity towards organic hydroperoxides. This activity is protective because it prevents organic hydroperoxides of phospholipids, fatty acids and DNA becoming engaged in free radical propagation reactions ultimately leading to the destruction of macromolecules during oxidative stress [2]. In addition to their enzymatic functions, GSTs have been found to act as regulatory proteins and serve in structural roles (S-crystallins). GSTs are involved in the sequestering and transport of exogenous hydrophobic compounds such as pesticides, herbicides and antibiotics; furthermore, they bind a large variety of endogenous compounds such as steroids, bilirubin, bile acids and ferriprotoporphyrin IX with varying affinities. This ‘ligandin’ activity may sequester toxic substances, effectively decreasing their concentration in the cell. The possible dual function of the GSTs, i.e. catalytic and storage/transport is interesting, since the non-substrate ligands can also act as inhibitors of enzymatic activity (for recent reviews [3, 4, 5, 6, 7 and 8]). The roles of the GSTs in the regulation of stress response, detoxification of lipid peroxidation products, the sequestration of potentially toxic compounds and, furthermore in drug resistance [9] are especially important considerations within a parasitic context. A blast search of the genome sequencing database of P. falciparum using GSTs from different classes identified the Pf-gst1 gene. To obtain the start methionine, the Pf-GST1 sequence was compared to GSTs from the alpha and the pi-class, followed by the design of primers and PCR using both P. falciparum genomic DNA and cDNA. Only one intron was found close to the 5′ end of the gene. The position and size of the intron is in full agreement with the genomic database. The exon/intron boundary lies in a distinct loop region following the first β-sheet. The position of the intron correlates with that of the first intron of the Onchocerca volvulus GST2 [10], an enzyme that has 31% amino acid identity to the Pf-GST1. The Pf-gst1 gene consists of 815 bp and is located on chromosome 14 (chr14_1; methionine at 802 916 bp and stop codon at 803 731 bp). Northern blot demonstrates that the Pf-GST1 is transcribed as a single 2.3 kb-transcript, indicating that the major portion of the transcript is non-coding (data not shown). In order to determine whether Pf-GST1 cDNA encodes a functional protein, it was expressed in a procaryotic expression system. SDS-PAGE analysis of the purified soluble enzyme revealed a protein of about 25 kDa (Fig. 1a, lane 2), consistent with the calculated molecular mass of the deduced amino acid sequence (24.8 kDa). The purification of rPf-GST1 resulted in 20–30 mg rPf-GST1 from a 1.5-liter culture. The recombinant enzyme was purified using affinity chromatography followed by gelfiltration. Affinity-purified rPf-GST1 was frozen at −20 °C for up to 14 days, where it was found to be stable. Around 30% of the original enzymatic activity was lost when the enzyme was precipitated with ammonium sulphate. Following precipitation, the enzyme was stable at 4 °C for 1 month. The molecular mass of the active recombinant Pf-GST1 was determined to be around 50 kDa by gelfiltration, indicating that the protein forms a dimer. Western blot analysis of P. falciparum extract using polyclonal anti-Pf-GST1 antibodies demonstrated that the native enzyme has the same size (Fig. 1b). Most GSTs described so far form dimers, with subunit molecular mass of around 25,000 [4]. Following gelfiltration the enzyme became conformationally unstable and the enzymatic activity decreased substantially. Instability was overcome by the addition of 5 mM GSH to the buffers. en
dc.format.extent 2 en
dc.language.iso eng
dc.relation.ispartof Molecular and Biochemical Parasitology en
dc.title The glutathione S-transferase from Plasmodium falciparum en
dc.type Text en
dc.type.publicationtype Article (Journal) en
dc.identifier.doi http://dx.doi.org/10.1016/S0166-6851(02)00160-3
dc.contributor.institution Institute of Biological, Environmental and Rural Sciences en
dc.description.status Peer reviewed en


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