Purification, characterization and kinetic properties of the multifunctional thioredoxin-glutathione reductase from Taenia crassiceps metacestode (cysticerci)
Por:
Rendón J.L., Del Arenal I.P., Guevara-Flores A., Uribe A., Plancarte A., Mendoza-Hernández G.
Publicada:
1 ene 2004
Resumen:
The multifunctional enzyme thioredoxin-glutathione reductase (TGR) was purified to homogeneity from the soluble fraction of Taenia crassiceps metacestode (cysticerci). Specific activities of 17.5 and 4.7Umg-1 were obtained with Plasmodium falciparum thioredoxin and GSSG, respectively, at pH 7.75. Under the same conditions, Km values of 17, 15, and 3µM were respectively calculated for thioredoxin, GSSG and NADPH. The k cat/Km ratio of T. crassiceps TGR for both thioredoxin and GSSG falls in the range observed for typical thioredoxin reductases and glutathione reductases. Purified enzyme also showed glutaredoxin activity, with a specific activity of 19.2Umg-1 with hydroxyethyl disulfide as substrate. Both thioredoxin and GSSG disulfide reductase activities were fully inhibited by nanomolar concentrations of the gold compound auranofin, supporting the existence of an essential selenocysteine residue. Relative molecular mass of native enzyme was 136,000±3000, while the corresponding value per subunit, obtained under denaturing conditions, was 66,000±1000. These results suggest TGR exists as a dimeric protein. Isoelectric point of the enzyme was at pH 5.2. Moderate or high concentrations of GSSG, but neither thioredoxin nor NADPH, resulted in a markedly hysteretic kinetic, characterized by a lag time before the steady state velocity was reached. The magnitude of the lag time was dependent on GSSG and enzyme concentration. Preincubation of the enzyme with micromolar concentrations of GSH or DTT abolished the hysteresis, suggesting that a thiol-disulfide exchange mechanism is involved. © 2003 Elsevier B.V. All rights reserved.
Filiaciones:
Rendón J.L.:
Departamento de Bioquímica, Facultad de Medicina, Univ. Nac. Auton. de Mex., Apartado Postal 70-159, México 04510, D.F., Mexico
Del Arenal I.P.:
Departamento de Bioquímica, Facultad de Medicina, Univ. Nac. Auton. de Mex., Apartado Postal 70-159, México 04510, D.F., Mexico
Guevara-Flores A.:
Departamento de Bioquímica, Facultad de Medicina, Univ. Nac. Auton. de Mex., Apartado Postal 70-159, México 04510, D.F., Mexico
Uribe A.:
Departamento de Bioquímica, Facultad de Medicina, Univ. Nac. Auton. de Mex., Apartado Postal 70-159, México 04510, D.F., Mexico
Plancarte A.:
Depto. de Microbiol. y Parasitol., Facultad de Medicina, Univ. Nac. Auton. de Mex., México 04510, D.F., Mexico
Mendoza-Hernández G.:
Departamento de Bioquímica, Facultad de Medicina, Univ. Nac. Auton. de Mex., Apartado Postal 70-159, México 04510, D.F., Mexico
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