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PubMed1993

In vitro activation of hepatic glutathione reductase from mice by lobenzarit disodium.

Glutathione reductase activity from mice liver is significantly enhanced by lobenzarit disodium at concentrations between 0.3 and 1.5 mM. A maximum activation of almost 30% is achieved at a drug concentration of 0.9 mM. Similar results were observed with glutathione reductase from human leukocytes, but not with the enzyme from yeast. By preincubation with the enzyme from mice liver, lobenzarit also proved to prevent, at least partially, the immediate inhibition caused by the well-known thiol-reacting agents, thus indicating a protecting effect on the catalytically important thiol residue of the enzyme. The results here obtained explain in part the recently found hepatoprotective effect of lobenzarit disodium against acute liver toxicity induced by acetaminophen in mice.

Animals↗

PubMed1991

Hysteretic behaviour and GSSG substrate inhibition shown by glutathione reductase from Phycomyces blakesleeanus.

Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.

Glutathione↗

PubMed1983

The effect of BCNU (carmustine) on tissue glutathione reductase activity.

A single, 50 mg/kg i.p. dose of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) to mice significantly inhibited glutathione reductase activity in liver within 10 min, and in lung and heart within 30 min. Maximal inhibition was reached at 4 h in all tissues and glutathione reductase activity remained significantly depressed for 48 h in liver and for 96 h in lung and heart. BCNU doses of less than 25 mg/kg had no effect on glutathione reductase activity. Increasing the BCNU dose to 100 mg/kg caused greater inhibition of glutathione reductase activity in all tissues. In spite of significantly decreased glutathione reductase activity, reduced glutathione (GSH) levels were unaffected in all tissues both 1 and 24 h after 50 mg/kg BNCU. These results suggest that lower than normal glutathione reductase activity is sufficient to maintain GSH levels. These results also demonstrate that BCNU inhibits tissue glutathione reductase activity for several days, but only at doses significantly greater than previously reported.

Animals↗

PubMed1989

Dimer-tetramer equilibrium of glutathione reductase from the cyanobacterium Spirulina maxima.

Glutathione reductase [NAD(P)H:GSSG oxidoreductase; EC 1.6.4.2] from cyanobacterium Spirulina maxima exists as an equilibrium system between a dimer (S20,W = 5.96) and a tetramer (S20,W = 8.49) which has a very slow interconversion rate at neutral pH. Our results showed that the apparent dissociation constant (kd) was 4.61 X 10(-7) M. The proportion of both forms at pH 7.0 did not alter at either 4 or 25 degrees C. However, electrophoretic analysis at various pH values showed that at 25 degrees C a gradual transition takes place between oligomers with an apparent pKa of 7.55. When dimers aggregate to form tetramers, the reaction involves the uptake of eight protons (K = 1.58 X 10(-64) M9). At pH 7.7, the equilibrium shifts completely from dimers-tetramers to dimers when temperature is increased, which would suggest that the dissociation is an endothermic process. Thermodynamic parameters obtained from the temperature study show that the dissociation of glutathione reductase is characterized by positive entropy and enthalpy changes. Neither NADPH nor GSSG have any effect on the dimer-tetramer equilibrium. Measurements of reductase activity indicate that the tetramer is almost certainly active, whereas the dimer is either less active or inactive.

Cyanobacteria↗

PubMed1996

Inactivation of yeast glutathione reductase by O-phthalaldehyde.

Yeast glutathione reductase was inactivated by the bifunctional reagent, o-phthalaldehyde. The initial rate of inactivation followed pseudo-first order kinetics. Fluorescence spectral properties of modified enzyme indicated the formation of an isoindole derivative from cysteine and lyaine residues present in close proximity as shown by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The fluorescence spectral studies with o-phthalaldehyde in the presence and absence of N-ethylmaleimide indicated that both the inhibitors react with the same cysteine residue, which is non-essential for enzyme activity. The coenzyme NADPH did not protect the enzyme against the o-phthalaldehyde reaction while oxidised glutathione prevented o-phthalaldehyde inactivation. This could be due to reaction of the amino group of glutathione with o-phthalaldehyde. Stoichiometry of the reaction showed that the formation of approximately 2 isoindole derivatives per subunit of glutathione reductase is accompanied by 75% loss of activity. The results suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.

Cysteine↗

PubMed1969

Glutathione reductase: stimulation in normal subjects by riboflavin supplementation.

Glutathione reductase of hemolyzates from clinically normal subjects is activated by the addition of flavin-adenine dinucleotide. One-half maximum stimulation could be achieved by approximately 0.02 micromolar flavin-adenine dinucleotide; prior addition of adenosine triphosphate, adenosine diphosphate, or adenosine mnonophosphate prevented activation. Stimulation of glutathione reductase activity of red cells of normal subjects occurred when they were given 5 milligrams of riboflavin daily for 8 days. The degree of stimulation in vitro by flavin-adenine dinucleotide and in vivo by riboflavin was inversely proportional to dietary intake of riboflavin. The variety of clinical disorders which have been associated with glutathione reductase deficiency may have, as a common denominator, abnormalities in flavin-adenine dinucleotide formation.

Adenine Nucleotides↗

PubMed1993

Folding of the four domains and dimerization are impaired by the Gly446-->Glu exchange in human glutathione reductase. Implications for the design of antiparasitic drugs.

Glutathione reductase (NADPH+GSSG+H+-->NADP(+) + 2GSH) is a homodimeric flavoenzyme of known geometry. Each subunit contains four well-defined domains and contributes essential residues to the active sites; consequently, the monomer is expected to be inactive. As part of our program to develop dimerization inhibitors of human glutathione reductase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, represent the tightest contact between the subunits [Karplus, P. A., & Schulz, G. E. (1987) J. Mol. Biol. 195, 701-729]. Wild-type human glutathione reductase and mutants of this protein were produced in plasmid-transformed Escherichia coli SG5 cells. Active enzyme species, namely, wild-type hGR, N-terminally truncated delta(1-15)hGR, and the point mutant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization. Inactive mutants such as G446E-hGR or the double mutants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorption chromatography. G446E/F447P-hGR was studied in detail. This mutant behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cys90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues thereof; a short half-life (< 4 min) in E. coli cells; and high susceptibility to trypsin in vitro. The results suggest that the sequence around G446 can control dimerization as well as domain folding. This is unexpected since the FAD-binding domain and the NADPH-binding domain occur in many different enzymes and have been regarded as autonomous folding units.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

Characterization of the inhibition of glutathione reductase and the recovery of enzyme activity in exponentially growing murine leukemia (L1210) cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea.

The inactivation of the enzyme glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied in exponentially growing murine leukemia cells. A 1-hr incubation with 1.6 +/- 0.2 microM BCNU resulted in a 50% inhibition of glutathione reductase, while 10 microM BCNU caused total inhibition of the enzyme. The time required for 50% inhibition of glutathione reductase by 10 microM BCNU was 7 min. The recovery of glutathione reductase activity was studied by incubating cells with 10 microM BCNU for 30 min to inhibit all glutathione reductase activity, washing the cells free of drug, and continuing the incubation in fresh medium. Fifty percent of the activity returned within 12 hr. Glutathione reductase activity recovered normally when cell growth and DNA synthesis were inhibited in the cells, but it failed to recover when protein synthesis was inhibited. Therefore, the inactivation of glutathione reductase appears irreversible, and the recovery of enzymatic activity is dependent on the synthesis of new protein. Continuous incubation with 19.8 +/- 0.4 microM BCNU resulted in a 50% inhibition of cell growth. A 1-hr incubation with 7.3 +/- 0.8 microM BCNU resulted in a 50% loss of viability as measured by a soft agar clonogenic assay. These experiments quantify the inhibition of glutathione reductase by BCNU and the recovery of enzyme activity in the context of the toxic effects of the compound. A clinically useful inhibitor of glutathione reductase will require a wider difference between the concentrations required for enzyme inhibition and cytotoxicity than BCNU provides.

Animals↗

PubMed1981

Glutathione reductase from human erythrocytes: amino-acid sequence of the structurally known FAD-binding domain.

Glutathione reductase (Mr 2 x 52 500), a flavoenzyme of known three-dimensional structure, catalyses the reduction of glutathione disulfide by NADPH. This paper describes the primary structure of the FAD-binding domain which ranges from AcAla-1 to Gly-157. The three CNBr-produced fragments (69, 10 and 80 residues) of the domain were fractionated further by enzymatic and chemical methods; isolated peptides were sequenced mainly by automatic solid-phase Edman degradation. The tryptic peptides were overlapped by chymotryptic peptides. A fragment which results from cleavage at the acid-labile bond between Asp-135 and Pro-136 supplied peptides for overlapping the CNBr-produced fragments. In addition, many peptides were ordered and overlapped by computerized comparison with a complete sequence guessed from the electron density map. With one exception the computer method and the chemical alignment gave the same results. The sequence data are discussed in the light of the secondary and tertiary structure (Schulz et al. (1978) Nature (Lond.) 273, 120--124]. The 17 N-terminal residues are not visible in the electron density map. Consequently our numbering scheme differs from that of Schulz et al. by approximately 20 residues. Acetylation of the N terminus and an unusual composition of the following residues may serve to protect the loose N-terminal section of the protein against proteolysis in situ. The four cysteinyl residues of the FAD domain are of special interest. Cys-2 at the tip of the N-terminal extension is likely to be involved in the aggregation behaviour of glutathione reductase. Cys-58 and Cys-63 (formerly Cys-41 and Cys-46) represent the enzyme's redox-active dithiol. Cys-90 with its location at the twofold axis forms a disulfide bridge with Cys-90 of the other peptide chain of the enzyme. This might be related to the fact that both peptide chains contribute to each of the two active centers. In view of the interchain disulfide bridge glutathione reductase should be regarded as a monomeric protein. The sequence of the FAD-binding domain was compared with the sequence of the NADPH-binding domain of glutathione reductase using a computer program. As discussed, the scarcity of sequence similarities does not argue against the assumption that the two nucleotide-binding domains of glutathione reductase originated by gene duplication. The pyrophosphate moiety of FAD binds to a part of the polypeptide chain which in geometric structure, in topology and in sequence resembles the phosphate loops of other nucleotide-binding proteins and of flavodoxin. Using the phosphate loop as a reference, the N-terminal sequence of five flavoproteins can be aligned. The results of Williams et al. on the sequence of lipoamide dehydrogenase (EC 1.6.4.3) and our data on glutathione reductase (EC 1.6.4.2) show clearly that these two mechanistically similar enzymes possess homologous structures.

Amino Acid Sequence↗

PubMed1996

Mitochondrial targeting of glutathione reductase requires a leader sequence.

Glutathione reductase (GR), which catalyzes the conversion of glutathione disulfide to glutathione, is encoded in nuclear DNA, but is active in cytoplasm and mitochondria. However, analyses of known protein and DNA sequences for human GR have not revealed a potential mitochondrial targeting signal (MTS). We generated two 5'-truncated GR clones, which resulted in omission of the N-terminal 5 or 10 amino acids, to disable a potential targeting signal, and generated two GR clones containing synthetic MTS cDNAs. Transfection of Chinese hamster ovary cells with the full length human GR cDNA or with the 5'-truncated clones increased cytosolic GR activities 6- to 14-fold, but increased mitochondrial activities less than 2-fold. In contrast, transfection with either of the GR clones containing MTS cDNAs increased GR activities in mitochondria more than 24-fold. We conclude that the existing protein and DNA sequences for human GR do not contain a MTS and that such a signal is needed for effective mitochondrial targeting.

Animals↗

PubMed1995

In vitro inactivation of yeast glutathione reductase by tetramethylthiuram disulphide.

Previous in vivo investigations have shown that glutathione reductase is one of the sites of action of the dithiocarbamate fungicide tetramethylthiuram disulphide (thiram) in the yeast Saccharomyces cerevisiae. The inactivation of glutathione reductase by thiram has now been demonstrated in vitro. This inactivation was time-dependent and occurred only with the enzyme in the reduced state and in the absence of glutathione. Since the turnover rate of the enzyme with thiram as a substrate was significantly higher than the rate of enzyme inactivation, it was suggested that more than one enzyme-inhibitor complex was involved in the reaction. Arguments supporting a covalent modification of glutathione reductase were further obtained by experiments carried out with [14C]thiram and gel filtration. A kinetic scheme for the inactivation is proposed and the relevance of the in vitro data to previous in vivo studies is discussed taking into consideration current concepts of glutathione reductase inactivation by affinity reagents.

Carbon Radioisotopes↗

PubMed1969