Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.47 (cysteine synthase)
 625 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:[The biosynthesis of glutathione in human erythrocytes (author's transl)]. 1 76

Gamma-Glutamyl-cysteine synthetase is inhibited by glutathione under conditions similar to those which prevail in vivo, thus strongly suggesting a physiologically significant feedback mechanism. Inhibition by glutathione, which is not allosteric, appears to involve the binding of glutathione to the glutamate site of the enzyme as well as to another enzyme site; the latter binding appears to require a sulfhydryl group since ophthalmic acid (gamma-glutamyl-alpha-aminobutyryl-glycine) is only a weak inhibitor. The finding that glutathione regulates its own synthesis by inhibiting synthesis of gamma-glutamyl-cysteine appears to explain observations on patients with 5-oxoprolinuria, who were shown to have a block in the gamma-glutamyl cycle consisting of a marked deficiency of glutathione synthetase and consequently of glutathione. These patients produce greater than normal amounts of gamma-glutamyl-cysteine, which is converted by the action of gamma-glutamyl cyclotransferase to 5-oxoproline; production of the latter compound exceeds the capacity of 5-oxoprolinase to convert it to glutamate. The apparent Km value for L-cysteine for gamma-glutamyl-cysteine synthetase (0.35 mM) is not far from intracellular concentrations of L-cysteine suggesting that the availability of L-cysteine may also play a role in the regulation of glutathione synthesis.
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PMID:Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. 111 10

The primary metabolic defect in 5-oxoprolinuria (pyroglutamic aciduria) is the lack of glutathione synthetase. The mechanism of the concomitant overproduction of 5-oxoproline was studied using cell-free extracts of erythrocytes from control individuals and from patients with 5-oxoprolinuria. Such extracts catalyzed the synthesis of 5-oxoproline from L-glutamate. Addition of ATP, Mg ions and alpha-aminobutyrate was needed for optimal activity. The conversion of glutamate to 5-oxoproline occurred in two steps, catalyzed by gamma-glutamyl-cysteine synthetase and gamma-glutamyl cyclotransferase, respectively. Extracts of erythrocytes from control subjects and patients with 5-oxoprolinuria had identical capacity to synthesize 5-oxoproline. The conversion of glutamate to 5-oxoproline was markedly inhibited by reduced glutathione, which exerted its effect on the gamma-glutamyl-cysteine synthetase step. The following mechanism is postulated for the overproduction of 5-oxoproline in 5-oxoprolinuria: the deficiency of glutathione synthetase causes a lack of glutathione which is an essential feed-back inhibitor in the initial step of its biosynthesis. Therefore gamma-glutamyl-cysteine is produced in excessive amounts and it is subsequently converted to 5-oxoproline (and cysteine) by gamma-glutamyl cyclotransferase. This overproduction of 5-oxoproline exceeds the capacity of the 5-oxoprolinase and 5-oxoproline accumulates in body fluids.
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PMID:On the mechanism of 5-oxoproline overproduction in 5-oxoprolinuria. 126 Oct 42

A number of enzyme systems are important in the protection of cells from chemical-induced oxidative damage. Little is known of the relative importance of these enzymes during postnatal development and its is possible that changes in their activity during this period may alter the susceptibility to toxic agents. This study investigated the activities of glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, gamma-glutamyl-cysteine synthetase and glutathione synthetase in the liver, lung and kidney of postnatal and adult mice. The first 3 postnatal weeks are characterized by marked changes in the activities of enzymes that protect against oxidative stress (glutathione peroxidase/reductase, catalase and superoxide dismutase). Overall, the activity of these enzymes suggests that the mouse has a higher level of protection against peroxides at various stages during this period but lower capacity to detoxify superoxide anions. The activities of the glutathione-synthetic enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) were significantly lower in the kidney of the postnatal mice, but the liver and lung had levels similar to those in the adult. Glutathione turnover in the liver of 2-week-old mice was not different from that in adults. The results indicate a complex pattern of development in the activities of detoxification enzyme systems during postnatal development.
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PMID:Postnatal development of enzyme activities associated with protection against oxidative stress in the mouse. 196 50

The two enzymes required for de novo glutathione synthesis, glutamyl cysteine synthetase and glutathione synthetase, have been demonstrated in hemolysates of human erythrocytes. Glutamyl cysteine synthetase requires glutamic acid, cysteine, adenosine triphosphate (ATP), and magnesium ions to form gamma-glutamyl cysteine. The activity of this enzyme in hemolysates from 25 normal subjects was 0.43+/-0.04 mumole glutamyl cysteine formed per g hemoglobin per min. Glutathione synthetase requires gamma-glutamyl cysteine, glycine, ATP, and magnesium ions to form glutathione. The activity of this enzyme in hemolysates from 25 normal subjects was 0.19+/-0.03 mumole glutathione formed per g hemoglobin per min. Glutathione synthetase also catalyzes an exchange reaction between glycine and glutathione, but this reaction is not significant under the conditions used for assay of hemolysates. The capacity for erythrocytes to synthesize glutathione exceeds the rate of glutathione turnover by 150-fold, indicating that there is considerable reserve capacity for glutathione synthesis. A patient with erythrocyte glutathione synthetase deficiency has been described. The inability of patients' extracts to synthesize glutathione is corrected by the addition of pure glutathione synthetase, indicating that there is no inhibitor in the patients' erythrocytes.
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PMID:Glutathione biosynthesis in human erythrocytes. I. Identification of the enzymes of glutathione synthesis in hemolysates. 554 17

Two brothers, aged 16 and 11 years, had recurrent episodes of vomiting, diarrhoea and abdominal pain, starting in infancy. In spite of extensive investigations no cause of their enterocolitis could be established. After several years symptomatic treatment was discontinued without any recurrence of symptoms. Their father and several paternal relatives have had kidney stones. Both boys developed urolithiasis and an oxalate-containing stone was removed from the elder brother's kidney. He had no hypercalciuria. His glomerular and tubular function tests were normal. Gas chromatography of urine from both brothers revealed massive excretion of L-5-oxoproline (pyroglutamic acid). Glutathione levels in erythrocytes of both patients were normal. The activities of enzymes of the gamma-glutamyl cycle were analysed in erythrocytes, leukocytes and cultured skin fibroblasts. The level of glutathione synthetase was normal, as was the affinity of this enzyme for its substrate gamma-glutamyl-cysteine. Feedback inhibition of gamma-glutamyl-cysteine synthetase by glutathione was also normal. Both patients had a specific deficiency of 5-oxoprolinase, the activity of which was 2-4% of that of control subjects. Their parents had intermediate 5-oxoprolinase activities in fibroblasts, indicating a recessive mode of inheritance. Thus, 5-oxoprolinuria in these two patients was due to a lack of 5-oxoprolinase, i.e., a new inborn error in the gamma-glutamyl cycle.
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PMID:5-oxoprolinuria due to hereditary 5-oxoprolinase deficiency in two brothers--a new inborn error of the gamma-glutamyl cycle. 611 26

A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21.8 +/- 2.2, 36.5 +/- 4.1 and 38.6 +/- 2.8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0.52 +/- 0.04, 2.25 +/- 0.67 and 3.35 +/- 0.65 nmol min-1 mg-1 protein, respectively. When gamma-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0.19 +/- 0.08 and 1.54 +/- 0.76 nmol-1 min mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs. 843 34

Activities of enzymes that protect the retina from reactive oxygen species were investigated in experimentally diabetic rats and experimentally galactosemic rats, two animal models known to develop vascular lesions consistent with diabetic retinopathy. Diabetes or experimental galactosemia of 2 months duration significantly decreased the activities of glutathione reductase and glutathione peroxidase in the retina while having no effect on the glutathione synthesizing enzymes glutathione synthetase and gamma-glutamyl cysteine synthetase. Activities of two other important antioxidant defense enzymes-superoxide dismutase (SOD) and catalase-also were decreased (by more than 25%) in retinas of diabetic rats and galactosemic rats. Administration of supplemental antioxidants, vitamins C and E, for the 2 months prevented the diabetes-induced impairment of antioxidant defense system in the retina. In experimentally galactosemic rats, the supplemental antioxidants were not as effective: SOD activity was normalized, but the enzymes of the glutathione redox cycle were only partly restored, and the subnormal catalase activity was unaffected. Diabetes or experimental galactosemia results in significant impairment of the antioxidant defense system in the retina, and exogenous antioxidant supplementation can help alleviate the subnormal activities of antioxidant defense enzymes.
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PMID:Abnormalities of retinal metabolism in diabetes or experimental galactosemia. IV. Antioxidant defense system. 901 21

The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively. The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh. E. coli BL21 was transformed by pTrc-gsh for expression of the related enzymes. Analysis of SDS-PAGE showed that the expected products were expressed. E. coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG. The expressed products were up to 25% of the total protein of the bacteria. Acetone-treated cells of the engineered strain could synthesize GSH efficiently.
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PMID:[Cloning and expression of the genes of glutathione synthetases]. 1133 Jan 98

Genomics projects have identified thousands of interesting new genes whose protein products need to be examined at the tissue, subcellular, and molecular levels. Furthermore, modern metabolic engineering requires accurate control of expression levels of multiple enzymes in complex pathways. The lack of specific immune reagents for characterization and monitoring of these numerous proteins limits all proteomic and metabolic engineering projects. We describe a rapid method of isolating monoclonal antibodies that required only sequence information from GenBank. We show that large synthetic peptides were highly immunogenic in mice and crude protein extracts were effective sources of antigen, thus eliminating the time-consuming step of purifying the target proteins for antibody production. A case study was made of the three-enzyme pathway for the synthesis of phytochelatins. Enzyme-linked immunosorbent assays and western blots with the recombinant proteins in crude extracts demonstrated that the monoclonal antibodies produced to synthetic peptides were highly specific for the different target proteins, gamma-glutamyl cysteine synthetase, glutathione synthetase, and phytochelatin synthase. Moreover, immunofluorescence localization studies with antibacterial gamma-glutamyl cysteine synthetase and antiglutathione synthetase antibodies demonstrated that these immune reagents reacted strongly with their respective target proteins in chemically fixed cells from transgenic plants. This approach enables research to progress rapidly from the genomic sequence of poorly characterized target genes, to protein-specific antibodies, to functional studies.
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PMID:Rapid isolation of monoclonal antibodies. Monitoring enzymes in the phytochelatin synthesis pathway. 1170 54


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