Two glutathione transferase isoforms isolated from juvenile cysts of Taenia crassiceps: identification, purification and characterization

• Published in: Journal of helminthology Vol. 92; no. 6; pp. 687 - 695
• Main Authors: Maldonado, G., Nava, G., Plancarte, A.
• Format: Journal Article
• Language: English
• Published: Cambridge, UK Cambridge University Press 01.11.2018
• Subjects: 1-Chloro-2,4-dinitrobenzene; Animals; Chromatography, Liquid; Cysts; Dinitrobenzene; Enzyme Inhibitors - analysis; Enzyme Stability; Enzymes; Ethics; Glutathione; Glutathione transferase; Glutathione Transferase - chemistry; Glutathione Transferase - isolation & purification; Glutathione Transferase - metabolism; Hydrogen-Ion Concentration; Indomethacin; Isoforms; Kinetics; Laboratory animals; Ligands; Lipids; Mass Spectrometry; Mass spectroscopy; Molecular Weight; Parasites; Parasitic diseases; Peptides; pH effects; Protein Isoforms - chemistry; Protein Isoforms - isolation & purification; Protein Isoforms - metabolism; Protein Multimerization; Proteins; Research Paper; Rodents; Signal transduction; Substrates; Taenia - enzymology; Taenia crassiceps; Temperature; Transferases; Water purification; Worms; United States > US
• ISSN: 0022-149X; 1475-2697
• DOI: 10.1017/S0022149X17000931

We identified and characterized the first two glutathione transferases (GSTs) isolated from juvenile cysts of Taenia crassiceps (EC 2.5.1.18). The two glutathione transferases (TcGST1 and TcGST2) were purified in a single-step protocol using glutathione (GSH)-sepharose chromatography in combination with a GSH gradient. The specific activities of TcGST1 and TcGST2 were 26 U mg−1 and 19 U mg−1, respectively, both at 25°C and pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates. The Km(CDNB) and Kcat(CDNB) values for TcGST1 and TcGST2 (0.86 μm and 62 s−1; 1.03 μm and 1.97 s−1, respectively) and Km(GSH) and Kcat(GSH) values for TcGST1 and TcGST2 (0.55 μm and 11.61 s−1; 0.3 μm and 32.3 s−1, respectively) were similar to those reported for mammalian and helminth GSTs. Mass spectrometry analysis showed that eight peptides from each of the two parasite transferases were a match for gi|29825896 glutathione transferase (Taenia solium), confirming that both enzymes are GSTs. The relative molecular masses were 54,000 ± 0.9 for the native enzymes and 27,500 ± 0.5 for the enzyme subunits. Thus, TcGST1 and TcGST2 are dimeric proteins. Optimal TcGST1 and TcGST2 activities were observed at pH 8.5 in the range of 20–55°C and pH 7.5 at 35–40°C, respectively. TcGST1 and TcGST2 were inhibited by cibacron blue (CB), bromosulphophthalein (BST), rose bengal (RB), indomethacin and haematin (Hm) with 50% inhibitory concentrations (IC50) in the μm range. TcGST1 was inhibited in a non-competitive manner by all tested inhibitors with the exception of indomethacin, which was uncompetitive. The discovery of these new GSTs facilitates the potential use of T. crassiceps as a model to investigate multifunctional GSTs.