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

Abstract

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.