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.

     

Significance of glutathione S-conjugate for glutathione metabolism in human erythrocytes

The significance of glutathione S-conjugate in the regulation of glutathione synthesis was studied using human erythrocyte gamma-glutamylcysteine synthetase. Feedback inhibition of the enzyme by reduced glutathione was released by the addition of the glutathione S-conjugate (S-2,4-dinitrophenyl glutathione). A half-maximal effect of glutathione S-conjugate on gamma-glutamylcysteine synthetase activity was obtained at approximately 1 microM; 50 microM glutathione S-conjugate in the presence of 10 mM glutathione actually increased the enzyme activity twofold above uninhibited levels. Glutathione S-conjugate had no effect on the enzyme activity in the absence of glutathione. When erythrocytes were exposed to the electrophile 1-chloro-2,4-dinitrobenzene, which forms a glutathione S-conjugate by the catalytic reaction of glutathione S-transferase, the level of glutathione synthesis increased. These data suggest that glutathione S-conjugate plays a role in stimulating the synthesis of glutathione.     

Primary stimuli of icosanoid release inhibit arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. Mechanism of action of hydrogen peroxide and methyl mercury in platelets

Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.     

On the mode of action and biochemical properties of anti-inflammatory drugs-II.

The inhibition of prostaglandin (PG) synthetase by nonsteroidal anti-inflammatory drugs (NSAID) is not well understood. Co-factors (glutathione and hydroquinone) are needed for maximum enzymatic activity in vitro, and we suggest that NSAID might inhibit PG synthetase partly by interfering with co-factor induced stimulation of the enzyme. This hypothesis was tested by: A) Examining the effect of glutathione, noradrenaline and hydroquinone on bull seminal vesicle (BSV) PG synthetase in vitro. The stimulatory effects were concentration-dependent. B) Three structurally distinct NSAID, indomethacin, aspirin and paracetamol, inhibited the stimulation by each co-factor in a concentration-related manner. Drug effectiveness also depended on the concentration of co-factor.     

Oxidative stresses induced the cystine transport activity in human erythrocytes

Cystine was transported into human erythrocytes in the presence of tertiary-butyl hydroperoxide (t-BH) or 1-chloro-2,4-dinitrobenzene (CDNB). The transport rate of cystine was dependent on the extracellular concentration of t-BH or CDNB, and on the incubation time. According to Dowex-1 column chromatography, the transported cystine was incorporated into fractions of glutathione disulfide (GSSG) and glutathione-S (GSH-S) conjugate. The transport of cystine was competitively inhibited by DL-homocystine and alanine. The inhibition rates by DL-homocystine and alanine were 75% and 68%, with similar Ki values of 0.7 mM and 0.6 mM, respectively. It is suggested that cystine transport is induced for glutathione synthesis when human erythrocytes are exposed to oxidative stresses. This transport system of cystine may serve as an emergency function in human erythrocytes.     

On the mode of action and biochemical properties of anti-inflammatory drugs-II

The inhibition of prostaglandin (PG) synthetase by nonsteroidal anti-inflammatory drugs (NSAID) is not well understood. Co-factors (glutathione and hydroquinone) are needed for maximum enzymatic activity $\text{in vitro}$, and we suggest that NSAID might inhibit PG synthetase partly by interfering with co-factor induced stimulation of the enzyme. This hypothesis was tested by:A) Examining the effect of glutathione, noradrenaline and hydroquinone on bull seminal vesicle (BSV) PG synthetase $\text{in vitro}$. The stimulatory effects were concentration-dependent.B) Three structurally distinct NSAID, indomethacin, aspirin and paracetamol, inhibited the stimulation by each co-factor in a concentration-related manner. Drug effectiveness also depended on the concentration of co-factor.     

Evidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytes

The effect of oxidized glutathione (GSSG) on the ATP-dependent transport of S-dinitrophenyl glutathione (Dnp-SG) by inside-out vesicles prepared from human erythrocytes and by intact erythrocytes has been studied. It is demonstrated that the transport of Dnp-SG is not inhibited by GSSG in either intact erythrocytes or in inside-out vesicles. These results suggest that Dnp-SG and GSSG are transported out of human erythrocytes by separate systems.     

The biosynthesis of glutathione in human erythrocytes (author's transl)]

The concentrations of glutathione precursors in human erythrocytes were investigated. 300muM glutamate, 375 muM glycine, and 10muM cysteine were found by automated amino acid analysis. The concentration of 2-aminobutyrate, the precursor of ophthalmic acid, was 15muM. The influence of the activities of endogenous or added glutamyl-cysteine synthetase and glutathione synthetase on the rate of glutathione biosynthesis was measured in membrane-free hemolysates under physiological conditions. The results show that the rate of the overall biosynthesis mainly depends on the formation of the dipeptide glutamyl-cysteine. The effect of glutathione precursor concentrations on the synthesis of the tripeptide was investigated at constant (endogenous) activities of the synthesizing enzymes. The rate was not enhanced by addition of glutamate and/or glycine unless cysteine or glutamyl-cysteine was also added. It is concluded that the concentration of cysteine limits the actual rate of the glutamyl-cysteine-synthetase reaction in vivo. No cysteine or bis(glutamyl)cystine was detected in human hemolysate; however, these disulfides were converted to glutathione. This indicates that erythrocytes have an appropriate system for their reduction, since the disulfides themselves are not substrates for the glutathione-synthesizing enzymes. Studies with intact human red cells indicate that the uptake of cysteine is the rate-determining step in the biosynthesis of glutathione.     

Cobalt regulation of heme synthesis and degradation in avian embryo liver cell culture

Inorganic cobalt was found to induce heme oxygenase activity in primary cultures of embryonic chick liver cells and to inhibit the induction of delta-aminolevulinate synthetase by the porphyrinogenic compounds allylisopropylacetamide, dicarbethoxy-1,4-dihydrocollidine, etiocholanolone, phenobarbital, Aroclor (R)1254, and secobarbital. Much smaller concentrations of Co2+ (5 muM) were required to inhibit delta-aminolevulinate synthetase than to induce heme oxygenase activity (50 muM). These effects of Co2+ on heme synthesis and heme degradation were potentiated by depletion of cellular glutathione content as a result of treatment with diethyl maleate. Cobalt inhibition of the induction of delta-aminolevulinate synthetase was of the same magnitude and probably involved the same mechanism as that produced by cobalt heme dimethyl ester and iron heme. The induction of heme oxygenase by cobalt could be blocked by cycloheximide. Plasma protein synthesis was not inhibited in the presence of concentrations of Co2+ which produced inhibition of delta-aminolevulinate synthetase or induction of heme oxygenase. Other metals such as Cd2+ and Cu2+ also inhibited the induction of delta-aminolevulinate synthetase by allylisopropylacetamide. These findings indicate that Co2+ can regulate heme metabolism directly in liver cells without intermediate actions on extrahepatic tissues. It is suggested that regulation of production of delta-aminolevulinate synthetase and heme oxygenase is mediated through the action of the metal ion rather than the metal in the form of a tetrapyrrole chelate.     

Treatment of glutathione synthetase deficient fibroblasts by inhibiting gamma-glutamyl transpeptidase activity with serine and borate.

Glutathione synthetase deficiency results in decreased cellular glutathione content and consequent overproduction of 5-oxoproline. L-serine in borate buffer inhibits γ-glutamyl transpeptidase, the major catabolic enzyme for glutathione. Treatment of glutathione synthetase deficient fibroblasts with 40mM serine and borate for 24 hours produced more than a 2-fold increase in cellular glutathione content. L-serine alone led to a smaller increase in glutathione level, and borate alone was without effect. On exposure to serine and borate, 5-oxoproline formation from L-glutamate was decreased to normal levels in glutathione synthetase deficient fibroblasts, presumably secondary to feedback inhibition of γ-glutamylcysteine synthetase by the increased intracellular glutathione concentration. Cellular free amino acid content was generally unaffected by such exposure although increases were observed in serine and phosphoserine. This model system suggests that γ-glutamyl transpeptidase inhibition may be a rational approach to alleviating the effects of glutathione synthetase deficiency.     

Effects of phenol compounds, glutathione analogues and a diuretic drug on glutathione S-transferase, glutathione reductase and glutathione peroxidase from canine erythrocytes.

1. Phenol compounds (ellagic acid, quercetin and purpurogallin), glutathione analogues (S-hexylglutathione and S-octylglutathione) and a diuretic drug (ethacrynic acid) were compared for their inhibitory effects on glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in the canine erythrocytes. 2. All these compounds inhibited GST activity; quercetin was found to be the most potent inhibitor. 3. Ellagic acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity; S-hexylglutathione and S-octylglutathione had no effect on GR and GSH-Px activities. 4. Quercetin and purpurogallin inhibited GST non-competitively toward glutathione, whereas ellagic acid showed a competitive inhibition. Ellagic acid and purpurogallin inhibited GR non-competitively toward oxidized glutathione.     

Inhibition of gamma-glutamylcysteine synthetase by cystamine: an approach to a therapy of 5-oxoprolinuria (pyroglutamic aciduria).

γ-Glutamylcysteine synthetase is strongly inhibited by cystamine; thus, 20 μM cystamine inhibited the activity by 50%. Inhibition is rapid and the inhibited enzyme is reactivated by dithiothreitol suggesting that cystamine reacts with an enzyme sulfhydryl group. Inhibition by cystamine is not prevented by MgATP, L-α-aminobutyrate, or L-glutamate suggesting that cystamine may not interact at the active site. Little or no inhibition was observed with N,N′-diacetyl cystamine, L-cystine, glutathione disulfide, 2-hydroxyethyl disulfide, and thioglycolate disulfide, whereas thiocholine disulfide produced moderate inhibition. Cystamine or an inhibitory analog of cystamine might be useful in the therapy of the disease 5-oxoprolinuria in which there is an overproduction of γ-glutamylcysteine.     

Relationship between cell age, glutathione and cation concentrations in sheep erythrocytes with a normal and a defective transport system for amino acids

Percoll density gradients were used to separate sheep erythrocytes according to cell age. Erythrocytes with low intracellular levels of glutathione (GSH) caused by an inherited deficiency of the System C amino acid transporter exhibited large age-related decreases in GSH and K+ content. In contrast, there was no age-related loss of intracellular GSH in normal sheep erythrocytes or in sheep erythrocytes with low GSH resulting from a diminished activity of gamma-glutamylcysteine synthetase. Loss of GSH from amino acid transport-deficient erythrocytes was paralleled by the progressive appearance of Heinz bodies in the cells, indicating an increased susceptibility to oxidative damage.     

GSH biosynthesis in glutathione deficient erythrocytes from Finnish landrace and Tasmanian merino sheep.

1. The maximum activities of the enzymes for the biosynthesis of GSH (gamma-glutamyl-cysteine synthetase and GSH synthetase) have been assayed in high GSH and low GSH erythrocytes from Tasmanian Merino and Finnish Landrace sheep. 2. For the Merinos, the activities (mumol product/g haemoglobin per min +/- S.E.M. (n)) in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.776 +/- 0.065 (11) and 0.375 +/- 0.063 (13); and GSH synthetase: 0.069 +/- 0.003 (11) and 0.066 +/- 0.002 (13). 3. For the Finnish Landrace sheep the activities in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.595 +/- 0.063 (12) and 0.555 +/- 0.033 (10) and gamma-glutamyl-cysteine synthetase: 0.073 +/- 0.002 (12) and 0.070 +/- 0.002 (10). 4. gamma-Glutamyl-cysteine synthetase was markedly inhibited by physiological GSH concentrations. No evidence was found for the presence of an inhibitor of GSH biosynthesis (other than GSH) in low GSH erythrocytes from Finnish Landrace sheep. 5. Although for the Merinos the low GSH trait can be explained in terms of a diminished activity of gamma-glutamyl-cysteine synthetase, no such explanation is tenable for the Finnish Landrace sheep.     

Modulation of rat liver S-adenosylmethionine synthetase activity by glutathione.

Rat liver S-adenosylmethionine (AdoMet) synthetase appears as high-M(r) (tetramer) and low-M(r) (dimer) forms. Both are inhibited in the presence of GSSG at pH 8. The calculated Ki values are 2.14 and 4.03 mM for the high- and low-M(r) forms, respectively. No effect on enzyme activity was observed in the presence of GSH, but modulation of inhibition by GSSG can be obtained by addition of GSH. At a total glutathione concentration (GSH + GSSG) of 10 mM, a KOX of 1.74 was calculated for the high-M(r) form, whereas this constant was 2.85 for the low-M(r) AdoMet synthetase. No incorporation of [35S]GSSG was observed in either of the enzyme forms, and inhibition of enzyme activity was correlated with dissociation of both AdoMet synthetases to a monomer. The data obtained in the presence of GSSG seem to suggest that oxidation leads to the formation of an intrasubunit disulfide. The possible regulation of AdoMet synthetase activity by the GSH/GSSG ratio is discussed, as well as its in vivo significance.     

Relationship between cell age,glutathione and cation concentrations in sheep erythrocytes with a normal and defective transport system for amino acids

Percoll density gradients were used to separate sheep erythrocytes according to cell age. Erythrocytes with low intracellular levels of glutathione (GSH) caused by an inherited deficiency of the System C amino acid transporter exhibited large age-realted decreases in GSH and K⁺ content. In contrast, there was no age-related loss of intracellular GSH in normal sheep erythrocytes or in sheep erythrocytes with low GSH resulting from a diminished activity of γ-glutamylcysteine synthetase. Loss of GSH from amino acid transport-deficient erythrocytes was parallel by the progressive appearance of Heinz bodies in the cells, indicating an increased susceptibility to oxidative damage.     

Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells.

Clotrimazole has been shown to have potent anti-malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself. Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter. This paper shows that transport of the model substrate, 3 microm dinitrophenyl S-glutathione, across erythrocyte membranes is inhibited by multidrug resistance-associated protein 1 (MRP1)-specific antibody, QCRL-3, strongly suggesting that the high affinity transport is mediated by MRP1. The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1. In both erythrocytes and MRP1-expressing tumour cells, MRP1-associated transport is inhibited by clotrimazole over the range 2-20 microm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1-expressing human tumour cells. It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells. These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti-malarial action.     

λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization

λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular mass (M_r) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg²⁺. With 0.2mM Cd²⁺ or Zn²⁺, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity and to dissociation into subunits (M_r 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be effective inhibitors of the plant enzyme too. The apparent K_m values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione (K_i=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm. Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48% of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the plant cell. Dedicated to Professor A. Prison on the occasion of his 80th birthday     

IN VIVO INHIBITION OF γ-GLUTAMYLCYSTEINE SYNTHETASE BY l-METHIONINE-RS-SULFOXIMINE; INFLUENCE ON INTERMEDIATES OF THE γ-GLUTAMYL CYCLE

—The inhibition of γ-glutamylcysteine synthetase and its influence on the concentration of intermediates associated with the metabolism of glutathione was studied in mice receiving methionine sulfoximine, a convulsant agent. The activity of the enzyme decreased significantly in the liver and kidney 1-4 h after administration of methionine sulfoximine; the activity of the enzyme in the brain was unchanged after 1 and 2 h but decreased significantly after 4 h. There was a rapid and sharp decrease in the concentration of glutathione in the kidney and a slower decrease in the liver. Brain glutathione concentrations were unaffected. Methionine sulfoximine in vivo, inhibited the synthesis of l -γ-glutamyl-l -α-aminobutyrate after administration of l -α-aminobutyrate, a reaction catalyzed by γ-glutamylcysteine synthetase. The inhibitor also lowered the concentration of pyrrolidone carboxylate in mouse tissues and prevented the accumulation of this intermediate after administration of l -α-aminobutyrate. The results show that methionine sulfoximine in vivo affects the metabolism of glutathione and that this action may contribute to its convulsive properties.     

Regulation of Glutathione Synthesis in Suspension Cultures of Parsley and Tobacco*

Experiments in vitro have shown that γ-EC synthesis, the first step in GSH formation, is subject to feedback inhibition by physiological GSH concentrations. In order to evaluate the role of this feedback inhibition on γ-EC synthetase in vivo GSH synthesis was modulated in suspension cultures of P. crispum and N. tabacum by administration of cadmium. The alterations in the thiol contents were measured and in addition the effect of Cd exposure on γ-EC synthetase (E.C. 6.3.2.2) and GSH synthetase (E.C. 6.3.2.3) was studied. Decreasing cellular GSH concentrations by cadmium induced PC synthesis caused 7–10 fold increase in the rate of glutathione synthesis as measured by the accumulation of (γ-EC)_nG. This increase was not linked to an increase in extractable activities of γ-EC- or GSH synthetase in parsley. In tobacco the activities of γ-EC- and GSH synthetase increased by a factor of 1.6 and 1.8, respectively, after 3 d of Cd exposure. In both species the exposure to Cd resulted in an increased cellular γ-EC content that reached a plateau within 24 h, and in a doubling of the cysteine content. In vitro experiments showed that GSH synthetase activity is inhibited by cadmium concentrations that have no effect on γ-EC synthetase activity. This may explain the accumulation of γ-EC in Cd exposed cells. Incubation with 0.25 mM cysteine did not effect the γ-EC- and GSH content in tobacco cells. In parsley the cellular GSH content increased threefold and the y-EC content twofold and stayed constant thereafter at the elevated levels. Taken together the results show that GSH synthesis in vivo is controlled by feedback inhibition as well as by the supply with cysteine. In the latter case the feedback inhibition may act as a kind of safety valve and prevent the accumulation of unphysiological GSH concentrations if the supply of cysteine is too large.