Molecular & Biochemical Parasitology 133 (2004) 61–69 Purification, characterization and kinetic properties of the multifunctional thioredoxin-glutathione reductase from Taenia crassiceps metacestode (cysticerci) Juan L. Rendón a,∗ , Irene P. del Arenal a , Alberto Guevara-Flores a , Aida Uribe a , Agust´ ın Plancarte b , Guillermo Mendoza-Hernández a a Departamento de Bioqu´ ımica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado Postal 70-159, México 04510, D.F., México b Departamento de Microbiolog´ ıa y Parasitolog´ ıa, Facultad de Medicina, Universidad Nacional Autónoma de México, México 04510, D.F., México Received 4 June 2003; accepted 10 September 2003 Abstract 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, K m values of 17, 15, and 3 M were respectively calculated for thioredoxin, GSSG and NADPH. The k cat /K m 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. © 2003 Elsevier B.V. All rights reserved. Keywords: Thioredoxin; Glutathione; Cysticerci; Cestode; Hysteresis 1. Introduction Glutathione (GSH) and thioredoxin (Trx) constitute the most important cellular thiols, which participate in a variety of processes [1,2]. Due to its relatively high concentration, GSH is the main contributor to the maintenance of a proper redox environment [3], also acting as a substrate for a va- riety of enzymes involved in the defense against reactive oxygen species [4,5]. Thioredoxin plays a more versatile role in the cell physiology, regulating the function of a number of protein factors and serving as an electron source for deoxyribonucleotide synthesis [6,7]. In all these events, Abbreviations: Trx, thioredoxin; Glrx, glutredoxin; TrxR, thioredoxin reductase; GR, glutathione reductase; HED, 2-hydroxyethyl disulfide; DTNB, 5,5 ′ -dithio-bis(2-nitrobenzoic acid) ∗ Corresponding author. Fax: +55-5-6162419. E-mail address: jrendon@bq.unam.mx (J.L. Rend´ on). the thiol groups of both GSH and Trx are continuously oxi- dized, making necessary the existence of enzymatic systems involved in the regeneration of the reduced forms of both compounds. In this regard, the participation of the flavoen- zymes glutathione reductase (EC 1.6.4.2 NAD[P]H:GSSG oxidoreductase) and thioredoxin reductase (EC 1.6.4.5 NADPH:Trx oxidoreductase) is critical to cell survival. These enzymes catalyze the NADPH-dependent reduction of the corresponding intramolecular disulfide bond. The structural features and kinetic properties of both glutathione reductase (GR) and thioredoxin reductase (TrxR) from a va- riety of organisms are known [8–13]. With few exceptions, GR is a homodimeric protein with subunits of about 55 kDa [8–10]. Its enzymatic activity is dependent on the presence of a redox active disulfide bond located in the interface between subunits [14]. TrxR, on the other hand, shows an interesting structural duality. While the enzyme from bacte- ria, fungi, plants and Entamoeba is represented by a dimeric 0166-6851/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2003.09.003