Purification, characterization and kinetic properties of the multifunctional thioredoxin-glutathione reductase from Taenia crassiceps metacestode (cysticerci).
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 4.7 U mg(-1) were obtained with Plasmodium falciparum thioredoxin and GSSG, respectively, at pH 7.75. Under the same conditions, Km values of 17, 15, and 3 microM were respectively calculated for thioredoxin, GSSG and NADPH. The kcat/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 19.2 U mg(-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.
Rendón
Juan L
JL
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado Postal 70-159, México 04510, D.F., México. jrendon@bq.unam.mx
del Arenal
Irene P
IP
Guevara-Flores
Alberto
A
Uribe
Aida
A
Plancarte
Agustín
A
Mendoza-Hernández
Guillermo
G
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003552:Cysticercus / Q000201:enzymology*; D019281:Dimerization; D005590:Fractional Precipitation; D005978:Glutathione / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry; D017279:Selenocysteine / Q000032:analysis; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013621:Taenia / Q000201:enzymology*; D013879:Thioredoxins / Q000378:metabolism
D009097:Multienzyme Complexes; D021122:Protein Subunits; D017279:Selenocysteine; D013879:Thioredoxins; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C466433:thioredoxin glutathione reductase; D005978:Glutathione; D000645:Ammonium Sulfate