Characterization of Glutathione S-Transferase of Taenia solium

Abstract

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.

Purification, characterization and kinetic properties of the Taenia solium glutathione S -transferase isoform 26.5�kDa

“…Purification of the T. solium GST activity fraction (SGSTF) was carried out by the technique described by Vibanco-Pe´rez et al (1999), with some modifications. Briefly, 2.5 g of parasites were suspended at a 1:10 ratio (w/v) in 0.01 M sodium phosphate buffer containing 0.15 M NaCl, pH 7.4 (PBS) and disrupted in a Polytron homogenizer (Brikmann Instruments, Westbury, N.Y.) (1,500 rpm).…”

Characterization of a recombinant mu-class glutathione S-transferase from Taenia solium

“…The fraction was composed of one major band observed in a 12% SDS-PAGE (Vibanco-Pe´rez et al 1999). However, when modifications were made to the purification process, we obtained a SGST fraction that showed two clear bands in a 5-22% gradient SDS-PAGE.…”

“…Purification of GST enzymes and protein sequencing A larval T. solium GST fraction (SGSTF) was purified as previously described (Vibanco-Pe´rez et al 1999), with minor modifications. In brief, Taenia crude extract was quickly filtered through a GSH-sepharose column (2.0·2.5 cm) (Pharmacia, Uppsala, Sweden) working at 50% of the capacity of the column.…”

“…We previously reported that the administration of 10 lg SGSTF with Freund's adjuvant was capable of reducing 74% of the parasite burden for at least 4 weeks in a T. crassiceps murine cysticercosis model (Vibanco-Pe´rez et al 1999). Here, after testing several combinations of antigen dose and adjuvant in the same murine model, we found that the most effective combination in reducing the parasites was immunization with 5 lg of each antigen using saponine as adjuvant.…”

“…Glutathione S-transferase activity was measured spectrophotometrically at 25°C as previously described (Vibanco-Pe´rez et al 1999) using CDNB, 1, 2-epoxy-3-(p-nitrophenoxy) propane, bromosulfophthalein, 1,2-dichloro-4-nitrobenzene, hexa-2,4-dienal, trans-non-2-enal (Aldrich, Milwaukee, Wis., USA), ethacrynic acid, glutathione reductase, GSH, H 2 O 2 and cumene-hydroperoxide (Sigma, St. Louis. Mo., USA).…”