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Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase

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Abstract

Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu2+ and Cu+ ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF–MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.

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Authors and Affiliations

  1. Department of Biochemistry, Aichi Medical University School of Medicine, Yazako-Karimata 1-1, Nagakute, Aichi, 480-1195, Japan

    Keiko Murakami, Ryoko Tsubouchi & Masataka Yoshino

  2. Central Research Laboratory, Aichi Medical University School of Medicine, Yazako-Karimata 1-1, Nagakute, Aichi, 480-1195, Japan

    Minoru Fukayama

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  1. Keiko Murakami
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  2. Ryoko Tsubouchi
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  3. Minoru Fukayama
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  4. Masataka Yoshino
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Correspondence to Masataka Yoshino.

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Murakami, K., Tsubouchi, R., Fukayama, M. et al. Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase. Biometals 27, 551–558 (2014). https://doi.org/10.1007/s10534-014-9731-x

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  • DOI: https://doi.org/10.1007/s10534-014-9731-x

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