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NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase

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Abstract

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP+, NAD+ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30° C. Lower but significant protection and reactivation was also observed with NADP+ and NAD+. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O2 or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD+ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.

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Abbreviations

DTNB:

5,5′-dithiobis(2-nitrobenzoate)

EGTA:

Ethylenglycoltetraacetic Acid

TNB:

5-thio-2-nitrobenzoate

Trx:

Thioredoxin

Trx(SH)2:

Reduced Thioredoxin

Trx-S2:

Oxidized Thioredoxin

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Authors and Affiliations

  1. Departamento de Bioquimica y Biología Molecular (Unidad de Veterinaria), Universidad de Córdoba, Avda. de Medina Azahara s/n, 14071, Cördoba, Spain

    E. Martínez-Galisteo, C. García-Alfonso, C. Alicia Padilla, J. Antonio Bárcena & J. López-Barea

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  1. E. Martínez-Galisteo
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  2. C. García-Alfonso
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  3. C. Alicia Padilla
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  4. J. Antonio Bárcena
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  5. J. López-Barea
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Martínez-Galisteo, E., García-Alfonso, C., Padilla, C.A. et al. NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase. Mol Cell Biochem 109, 61–69 (1992). https://doi.org/10.1007/BF00230874

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  • DOI: https://doi.org/10.1007/BF00230874

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