Inhibition of human glutathione reductase by the nitrosourea drugs 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea and 1‐(2‐chloroethyl)‐3‐(2‐hydroxyethyl)‐1‐nitrosourea

Karplus, P. Andrew; Krauth‐Siegel, R. Luise; Schirmer, R. Heiner; Schulz, Georg E.

doi:10.1111/j.1432-1033.1988.tb13775.x

Cited by 74 publications

(55 citation statements)

References 19 publications

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“…For carmustine (which is also active against rodent malaria), the modification of GR has been shown to occur also in vivo (28,29). Indeed EH 2 (FAD)(SH) 2 and its complex with NADPH appear to be the predominant intracellular PfGR forms.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum

Böhme¹,

Arscott²,

Becker³

et al. 2000

Journal of Biological Chemistry

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The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG ؉ NADPH ؉ H ؉ 7 2 GSH ؉ NADP ؉ ). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative halfreaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E ox -EH 2 and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K eq from the Haldane relationship, 0.075, very similar to the K eq of the same reaction for the yeast enzyme ( (S-S)⅐NADP؉ and E(FAD)(SH) 2 ⅐NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.The glutathione disulfide/glutathione (GSSG/GSH) redox couple is present in most eukaryotic cells at millimolar concentrations (1). Major functions of GSH include the detoxification of reactive oxygen species by donation of reducing equivalents to glutathione peroxidase and glutathione S-transferase as well as enzyme regulation by thiol-disulfide interchange reactions (2). The flavoenzyme glutathione reductase (GR, 1 EC 1.6.4.2) catalyzes the reduction of GSSG to GSH and creates an intracellular GSH/GSSG ratio of 20 to 1000 depending on the respective metabolic conditions (3). Thus, glutathione reductase, a protein of 2 ϫ 55 kDa, plays a key role in cellular redox homeostasis. Both human GR (hGR) and Plasmodium falciparum GR (PfGR) are essential for the survival of the malarial parasite within the human erythrocyte (4, 5). The amino acid sequence identity between the two enzymes is 45% with two major features distinguishing them: (i) an insertion of 34 amino acids within the central domain (residues 318 -351) between the FAD binding motif and the highly conserved H8 helix (6); (ii) the differences in the amino acid residues lining the wall of the interface cavity, where only 8 out of 25 residues are conserved in PfGR. This cavity represents the binding site of many inhibitors including isoalloxazines, safranine, and menadione in human GR and could be the binding site of methylene blue in PfGR (7).Glutathione reductase belongs to the pyridine nucleotidedisulfide oxidoreductase family of homodimeric flavoenzymes that includes lipoamide dehydrogenase and thioredoxin reductase. Like other members of this family, each subunit of GR contains a disulfide and a flavin that are in redox contact. This ground state of the enzyme is ...

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Section: Discussionmentioning

confidence: 99%

Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum

Böhme¹,

Arscott²,

Becker³

et al. 2000

Journal of Biological Chemistry

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“…One day later, the cells were treated with graded concentrations of the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-l-nitrosourea (HCNU) (Frischer and Ahmad, 1977;Karplus et al, 1988) in combi- nation with or without 12-0-tetradecanoyl-phorbol 13-acetate (TPA). The transactivation was determined after another 36 h by CAT assay.…”

Section: Distinct Responses Of Nfkb and Ap-1 To Changes Of Intracellumentioning

confidence: 99%

Distinct effects of glutathione disulphide on the nuclear transcription factors κB and the activator protein‐1

Galter

Mihm

Dröge

1994

European Journal of Biochemistry

263

144

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Oxidative conditions potentiate the activation of the nuclear transcription factor KB (NFKB) and the activator protein-1 (AP-1) in intact cells, but inhibit their DNA binding activity in vitro. We now show that both the activation of NFKB and the inhibition of its DNA binding activity is modulated in intact cells by the physiological oxidant glutathione disulphide (GSSG). NFKB activation in human T lineage cells (Molt-4) by 12-0-tetradecanoyl-phorbol 13-acetate was inhibited by dithiothreitol, and this was partly reversed by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1 -nitrosourea (BCNU) or by hydrogen peroxide, indicating that GSSG may be required for NFKB activation. These effects of BCNU and hydrogen peroxide were not seen in glutathionedepleted cells. However, NFKB and AP-1 activation were potentiated by dithiothreitol if added to cell cultures 1 h after the phorbol ester, indicating that a shift of redox conditions may support optimal oxidative activation with minimal inhibition of DNA binding. The elevation of intracellular GSSG levels by BCNU before stimulation suppressed the chloramphenicol acetyltransferase expression dependent on NFKB but increased that dependent on AP-1. This selective suppression of NFKB was also demonstrable by electrophoretic mobility shift assays. In vitro, GSSG inhibited the DNA binding activity of NFKB more effectively than that of AP-1, while AP-3 was inhibited more effectively by oxidized thioredoxin.The nuclear transcription factor KB (NFKB) is involved in the inducible transcription of immunologically relevant genes including the genes of the interleukin-2 receptor achain, tumor necrosis factor-a, major histocompatibility complex antigens and c-fos (Leung and Nabel, 1988;Cross et al., 1989; reviewed by Ullmann et al., 1990). The NFKB protein is found in many different cell types including B and T lymphocytes, macrophages and monocytes (reviewed by Baeuerle and Baltimore, 1991). The transcriptional activation of NFKB-dependent genes is usually induced by dissociation of the inhibitory protein IKB from the NFKB heterodimer in the cytoplasm and the subsequent translocation of active NFKB into the nucleus. The Fos and Jun proteins are components of another family of nuclear transcription factors that have been implicated in the regulation of growth, differentiation, neuronal excitation and cellular stress in a number of different cell types (reviewed by Angel and Karin, 1991;. Fos and Jun form a heterodimeric complex that interacts with a DNA sequence known as the activator protein-1 (AP-1) binding site or TPA-responsive element (TRE; TPA = 12-0-tetradecanoyl-phorbol 13-acetate). NFKB and other related factors of the v-and c-Re1 oncoprotein family share a characteristic sequence motif with a cysteine and three arginine residues in the DNA binding region (Ghosh et al., 1990;Kieran et al., 1990;Kumar et al., 1992). The DNA binding region of Fos, Jun and several related transcription factors also contains a characteristic sequence with a cysteine and up to seven ar...

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“…Enzyme is inactivated by SH-reagents, e.g. N-ethylmaleimide strongly influenced GSSG-reduction but the effect on diaphorase activity is very slight 17,18 . If only quercetin could modify enzyme at the intermadiate state -GRH 2 than preincubation of GR with QU increases its inhibition effect and contrary inhibition efficiency of galangin could not be dependent on preincubation of enzyme with NADPH.…”

Section: Resultsmentioning

confidence: 99%

The Effect of Quercetin and Galangin on Glutathione Reductase

Paulíková¹,

Berczeliová²

2005

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub

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Quercetin and galangin can change the activity of glutathione reductase. Quercetin (a catechol structure in the B-ring) and galangin (any hydroxyl group in the B-ring) have different biological activities but, both possess high antioxidant abilities. Quercetin during the antioxidative action, is converted into an oxidized products (o-semiquinone and o-quinone), and subsequently glutathionyl adducts may be formed or SH-enzyme can be inhibited. We have tried to see whether inhibition of glutathione reductase (GR) can be influenced by preincubation of enzyme with NADPH (a creation of reduced form of enzyme, GRH 2 ) and whether diaphorase activity of the enzyme is decreased by these flavonoids. The results confirmed that quercetin inhibits GRH 2 and inhibition is reduced by addition of EDTA or N-acetylcysteine. Both of flavonoids have no effect on diaphorase activity of glutathione reductase and this enzyme could increase the production of free radicals by catalysis of reduction of o-quinone during action of quercetin in vivo.

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Inhibition of human glutathione reductase by the nitrosourea drugs 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea and 1‐(2‐chloroethyl)‐3‐(2‐hydroxyethyl)‐1‐nitrosourea

Cited by 74 publications

References 19 publications

Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum

Kinetic Characterization of Glutathione Reductase from the Malarial Parasite Plasmodium falciparum

Distinct effects of glutathione disulphide on the nuclear transcription factors κB and the activator protein‐1

The Effect of Quercetin and Galangin on Glutathione Reductase

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