Glutathione Synthetase Activity Research Articles

Vitamin E and red blood cell glutathione

High doses of orally administered vitamin E (1000 IU/day) have been given to ten normal volunteers. Ten control subjects received placebo. Red blood cell glutathione was significantly higher in treated subjects than in the controls (controls: 267.5 ± 15.7 μg/mL; treated: 374.8 ± 17.3 μg/mL). These findings could be explained by an increase of glutathione synthesis brought about by the stimulation of glutathione synthetase activity. An alternative possibility is a reduced utilization of glutathione for the detoxification of free radicals. These two mechanisms could be effective in counteracting the glutathione content feedback of the synthetizing enzymes.

Survival curves of glutathione synthetase deficient human fibroblasts: correlation between radiosensitivity in hypoxia and glutathione synthetase activity.

The role of intracellular non-protein bound sulphydryl compounds (NPSH), and in particular that of glutathione (GSH), in the response of cells to ionizing radiation under different O2 concentrations has been assessed using cell strains deficient in glutathione synthetase and exhibiting different NPSH levels. The cell strains used originated from patients with 5-oxoprolinuria and from their relatives (heterozygotes and proficient homozygotes). No correlation has been found between NPSH and GSH concentrations and radiosensitivity under oxic, aerobic and hypoxic conditions. However, a highly significant correlation has been observed between radiosensitivity under hypoxic conditions (and therefore the oxygen enhancement ratio) and the glutathione synthetase activity, suggesting that synthesis of GSH is required after irradiation. In order to explain our results we postulated, beside radical processes, the existence of a GSH-dependent enzymatic repair mechanism for N2 type damage. Hypoxic radio-sensitivity measured with survival curves would result from the interaction of both competition and biochemical repair processes.

Normal glutathione content and some related enzyme activities in the fetal erythrocytes.

Pure fetal blood was obtained by direct-vision fetoscopy from 66 fetuses at 17-24 weeks gestation. The concentration of GSH and the activities of the enzymes gamma-glutamylcysteine synthetase (GCS), glutathione synthetase (GS), glutathione reductase (GR) and glutathione peroxidase (GPx) were analysed by established techniques to find the normal ranges for this gestational age. The ranges were relatively narrow and could serve as reference values for the prenatal diagnosis of defects in the GSH metabolism of erythrocytes. The results were compared with those obtained from 38 normal adults and with published values on neonatal blood. In the case of GR a comparison was also made with maternal blood. In comparison with adults, fetal erythrocytes showed higher GSH concentration and GCS activity and lower GS and GPx activities. This pattern resembled that found in neonatal erythrocytes except for the GCS activity, which was higher in the fetal cells. Furthermore the differences between fetal and adult erythrocytes were more pronounced than those between neonatal and adult cells. The GR activity of fetal erythrocytes was also higher than that of either normal adult or maternal blood. This difference, however, was reduced to an insignificant level when the enzyme was activated in vitro by flavin adenine dinucleotide (FAD) because of a relatively low per cent activation of the GR in the fetal erythrocytes.

Glutathathione synthetase of bovine lens: anomalies of the enzyme-catalyzed formation of ophthalmic acid.

The activity of glutathione synthetase from bovine lens was examined as a functions of the concentration of L-gamma-glutamyl-L-alpha-aminobutyrate, the dipeptide substrate required in the formation of ophthalmic acid. Several significant anomalies of the glutathione synthetase-catalyzed formation of ophthalmic acid were found. Curvilinearity of double reciprocal plots occurred with this substrate; this curvilinearity shows substrate activation of the reaction which is likely a result of negative cooperativity. Both ATP4- and, to a lesser extent Mg2+ inhibited the reaction, whereas MgATP2- is the substrate; maximum activity occurred with 2 mM Mg2+ in excess of the concentration of added ATP. This investigation shows that it is necessary to establish a defined set of conditions for reporting enzyme activity and that the usual practice of using very large concentrations of Mg2+ relative to ATP, and 5- to 20-fold excess of the dipeptide will give less than optimum activity. The unit of enzyme activity is suggested to be that activity in ml using 2 mM ATP, 4 mM Mg2+, 30 mM glycine and 15 mM L-gamma-glutamyl-alpha-aminobutyrate, which results in the formation of 1 nmole/minute of ADP or P(i). In this study, 5'-AMP was for the first time, shown to be an inhibitor of the reaction with a K(i) of 0.9 mM.

Some properties of glutathione biosynthesis-deficient mutants of Escherichia coli B.

Mutants of Escherichia coli B that contain essentially no detectable glutathione were isolated. These mutants had a very low activity of gamma-glutamylcysteine synthetase or glutathione synthetase. No significant differences in growth in minimal medium were observed between the mutants and the parental strain. The mutants lacking gamma-glutamylcysteine synthetase activity were more susceptible to toxic compounds than either the parental strain or a glutathione synthetase-deficient strain. The mutants lacking gamma-glutamylcysteine synthetase activity were also susceptible to oxygen.

Biochemical investigations of biopsied brain tissue and autopsied organs from a patient with pyroglutamic acidemia (5-oxoprolinemia)

Biochemical investigations have been carried out in autopsied tissues from a patient with pyroglutamic acidemia who died recently from thrombo-embolism. Very low activities of glutathione synthetase and a high content of pyroglutamate were found in all tissues examined. Especially high levels of pyroglutamate were found in tissues possessing high activity of γ-glutamyl cyclotransferase, namely the kidney and the brain. In addition to reduced feed-back inhibition by glutathione on γ-glutamyl-cysteine synthetase, we suggest that an increased availability of free cysteine also may be an important factor in the overproduction of pyroglutamate in these patients. The glutathione content was markedly reduced in all regions of the patient's brain, being especially low in the cerebellar cortex, which was the region showing the most prominent pathological changes. The low content of glutamate, γ-aminobutyric acid and taurine in this region probably reflects neuronal losses. A frontal cortical biopsy from the patient obtained some weeks before his death contained markedly reduced amounts of glutathione (32% of the normal mean), but most other amino compounds were present in normal concentrations. The cerebral lesions found in our patient closely resembled those found in patients with chronic organic mercury intoxication (Minamata disease). It is likely that the cerebral damage occurring in our patient was caused by lack of the normal protection against oxidative damage. If progressive neurological damage appears in these patients, attempts at treatment with some thiol compound or other antioxidant capable of crossing the blood-brain barrier should be considered.

Induction and repair of single-strand DNA breaks after X-irradiation of human fibroblasts deficient in glutathione.

Using the unwinding technique in weak alkali, the induction and repair of DNA single-strand breaks was determined after aerobic and anerobic X-irradiation of human fibroblasts, obtained from a patient suffering from 5-oxoprolinuria, and from a clinically healthy control. The metabolic disorder associated with 5-oxprolinuria is a deficiency in glutathione synthetase activity resulting in a greatly reduced glutathione content in the cells. A small dose-modifying effect of oxygen (o.e.r. = 1.1) was found for these cells in comparison to an o.e.r. of 2.5 for control cells with normal glutathione content. No significant difference was found between the repair capacity of cells with normal and deficient glutathione content, and repair was nearly completed within 60 min of anoxic irradiation in each case. In contrast, after aerobic irradiation of glutathione-deficient cells repaired less than 70 per cent of the breaks during the same period. When the glutathione-deficient cells were incubated with either dithiothreitol or mercaptopropionylglycine directly after aerobic irradiation, almost complete repair was obtained within 60 Min. The data are interpreted as indicating that the repair mechanism for oxically and anoxically induced single-strand breaks is qualitatively different, and requires glutathione in the former case.

Glutathione production coupled with an ATP regeneration system

Escherichia coli cells possessing glutathione synthetase and acetate kinase activities were immobilized with carrageenan gel. To enhance the operational stability, immobilized cells were treated with hardening agent, glutaraldehyde in the presence of hexamethylenediamine. The continuous production of glutathione was investigated using the column packed with immobilized Escherichia coli cell preparations. Glutathione was continuously produced by this column in the presence of acetyl phosphate and the half-life of this column was calculated to be 8 days at the flow rate of S.V.=0.1 h−1 at 37°C.

Association of glutathione synthetase deficiency and diminished amino acid transport in yeast

Summary Previous studies have shown that yeast contains high concentrations of glutathione and the enzymes of the γ-glutamyl cycle, suggesting that this cycle may be operative in yeast. Present studies with two cultured strains of Saccharomyces cerevisiae (wild-type strain 1278b and mutant strain 2512c gap) show a difference in [14C] glycine uptake. The mutant shows a decreased level of glutathione and a decreased glutathione synthetase activity.

Effect of R-25788 on EPTC Metabolism in Corn(Zea mays)

R-25788(N,N-diallyl-2,2-dichloroacetamide) had no effect on14CO2evolution from carbonyl-14C-EPTC(S-ethyl dipropylthiocarbamate) treated corn(Zea maysL. ‘inbred Oh551’). Treatment with 1.0 ppmw R-25788 increased production of water-soluble and unextractable products from EPTC and increased the rate of disappearance of organic-soluble radioactivity. R-25788 increased levels of reduced and oxidized glutathione in corn roots. Pretreatment of corn in nutrient solution with R-25788 had no effect on glutathione synthetase activity in root tissue, but R-25788 at 5.0 and 500.0 nM showed an in vitro stimulation of the enzyme.

Erythrocyte glutathione synthetase in 5-oxoprolinuria: Kinetic studies of the mutant enzyme and detection of heterozygotes

The primary metabolic defect in 5-oxoprolinuria is a generalized deficiency of glutathione synthetase. The activity of this enzyme was determined in cellfree extracts of erythrocytes from patients with 5-oxoprolinuria, their parents and a sibling as well as from normal control individuals. The following activities (pkat/mg of hemoglobin) for glutathione synthetase were obtained: homozygotes mean 0.10 (range 0.07–0.12), heterozygotes mean 3.1 (range 2.8–3.7) and control individuals mean 6.1 (range 5.4–6.7). These results indicate that 5-oxoprolinuria, i.e. the defective gluthione synthetase gene(s), is transmitted by autosomal recessive inheritance. Studies of the kinetics of the low remaining activity of erythrocyte glutathione synthetase in patients with 5-oxoprolinuria failed to reveal defective affinity for glycine, γ-glutamyl-α-aminobutyrate, ATP and Mg 2+ ions. Furthermore, the pH optimum, time curves and temperature dependence for the mutant enzyme activity did not significantly differ from the corresponding parameters observed with normal enzyme.

Biochemical Studies of Erythrocytes in a Patient with Pyroglutamic Acidemia (5-Oxoprolinemia)

Abstract An adult with pyroglutamic acidemia was investigated biochemically in efforts to explain his overproduction of pyroglutamate and his neurologic disease. The patient's erythrocytes contained no detectable glutathione, and their glutathione synthetase activity was less than 2 per cent of normal. These cells were loaded with free amino acids, most of them in concentrations five to 100 times normal. The erythrocytes also contained the unusual compound methionine sulfoxide. In skeletal muscle, in which glutathione was only reduced to 3 per cent of normal, there was no amino acid accumulation. The patient's overproduction of pyroglutamate is probably caused by increased in vivo activity of γ-glutamyl-cysteine synthetase due to absence of normal feedback inhibition by glutathione, with a resulting abundance of substrates available for γ-glutamyl cyclotransferase. Although lack of glutathione is apparently tolerable in short-lived erythrocytes, it could be disastrous in non-renewable neurons. Glutathione...

The enzymes of glutathione synthesis in erythrocytes from gsh-high and gsh-low type australian merino sheep

1. 1. The activity of γ-glutamylcysteine synthetase and glutathione synthetase was determined in erythrocytes from Australian Merino sheep of different erythrocyte haemoglobin, potassium and GSH types. 2. 2. The activity of γ-glutamylcysteine synthetase was diminished in GSH-low type erythrocytes, but glutathione synthetase had a similar activity in erythrocytes of both GSH types. 3. 3. γ-Glutamylcysteine synthetase from erythrocytes of both GSH types was inhibited by physiological concentrations of GSH, but a greater inhibition of the enzyme from GSH-low type erythrocytes occurred at low GSH concentrations.

5-OXOPROLINURIA /PYROGLUTAMIC ACIDURIA/ AN INBORN ERROR OF GLUTATHIONE METABOLISM

Two siblings suffering from metabolic acidosis and hemolytic anemia excreted gram quantities of 5-oxoproline in the urine. In erythrocytes, cultured fibroblasts and placenta the levels of glutathione were markedly decreased. The same tissues contained decreased activity of glutathione synthetase, whereas the activities of other enzymes involved in the metabolism of glutathione were normal. Extracts of erythrocytes from the patients and control subjects catalyzed the synthesis of 5-oxoproline from glutamate, provided ATP, Mg ions and cysteine were added. This occurred in two steps: gamma-glutamyl-cysteine synthetase catalyzed the formation of gamma-glutamyl-cysteine, which was converted to 5-oxoproline and cysteine by gamma-glutamyl cyclotransferase. Reduced glutathione specifically inhibited gamma-glutamyl-cysteine syuthetase. The following molecular mechanism for 5-oxoprolinuria is proposed: The lack of glutathione synthetase leads to a deficiency of glutathione. The absence of this feedback inhibitor results in increased synthesis of gamma-glutamyl-cysteine which is converted to 5-oxoproline /and cysteine/. The overproduction of 5-oxoproline exceeds the capacity of the 5-oxoprolinase. Therefore 5-oxoproline accumulates in body fluids.

Enzymes of glutathione synthesis in patients with myeloproliferative disorders

The concentration of reduced glutathione (GSH) and activities of γ-glutamylcysteine synthetase and glutathione synthetase have been measured in red cells from normal donors and from patients suffering from those hematological diseases which are sometimes associated with increased GSH levels. The activity of glutathione synthetase was strongly correlated with red cell GSH levels. In contrast, the level of γ-glutamylcysteine synthetase was unrelated to the concentration of GSH. These findings suggest that the increased level of glutathione in red cells of patients with myeloproliferative syndromes may be due, at least in part, to increase in the activity of glutathione synthetase.

The effect of methylene blue and diaminodiphenysulfone on red cell reduced glutathione synthesis.

Abstract The feeding of diaminodiphenylsulfone (DDS) or injection of methylene blue (MeBI) into rabbits resulted in rapid increase of red cell GSH (reduced glutathione). An increase in the total glutathione level of rabbit red cells was produced in vitro by incubation with DDS or MeBI. These compounds were shown to stimulate the activity of glutathione synthetase both of rabbit and human red cells, but to have little or no effect on γ glutamylcysteine synthetase. It is suggested that the increase of glutathione levels found in humans and in experimental animals after the administration of such drugs is due to stimulation of glutathione synthesis by activation of red cell glutathione synthetase.