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

Published online by Cambridge University Press:  16 October 2017

G. Maldonado ,
G. Nava  and
A. Plancarte
G. Maldonado
Affiliation:
Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad Nacional Autónoma de Mexico, UNAM, Ciudad de México, 04510, México
G. Nava
Affiliation:
Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad Nacional Autónoma de Mexico, UNAM, Ciudad de México, 04510, México
A. Plancarte*
Affiliation:
Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad Nacional Autónoma de Mexico, UNAM, Ciudad de México, 04510, México
*
Author for correspondence: A. Plancarte, Fax: +52 55 56232384, E-mail: apc@unam.mx
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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.

Type
Research Paper
Information
Journal of Helminthology , Volume 92 , Issue 6 , November 2018 , pp. 687 - 695
Copyright
Copyright © Cambridge University Press 2017 

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Footnotes

†

These authors contributed equally to the work.

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