Dataset Information

Assay, purification, properties and mechanism of action of gamma-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis.

ABSTRACT: 1. An improved radioassay for glutathione synthetase and gamma-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver gamma-glutamylcysteine synthetase was purified 324-fold by saline-bicarbonate extraction, protamine sulphate precipitation, CM-cellulose and DEAE-cellulose column chromatography, and gel filtration. 3. Rat liver gamma-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver gamma-glutamylcysteine synthetase activity was inhibited by GSH and activated by glycine. These effects, which were not found in the enzyme from Xenopus laevis, may have a regulatory significance. 5. Isotope-exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetases. The enzyme from Xenopus laevis appears to follow a Bi Bi Uni Uni Ping Pong mechanism, with glutamyl-enzyme as intermediate before the addition of cysteine and the release of gamma-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.

PROVIDER: S-EPMC1177756 | BioStudies | 1973-01-01

REPOSITORIES: biostudies

ACCESS DATA

Similar Datasets

Project description:The treatment of rats with cis-platinum (cis-diamminedichloroplatinum) for 1, 3 or 7 days elicited vastly different responses in the liver and the kidney in activities of enzymes of haem-metabolism pathway and gamma-glutamyl-cycle enzymes. The differences resided in the magnitude, direction and the time course of responses. In general, the liver was by far less severely affected, and when a response was elicited, it displayed an earlier onset (1-3 days), with a return to normal at 7 days. In the kidney, however, the effects were notable after 3 days of treatment, and became more pronounced at 7 days. Specifically, the activity of 5-aminolaevulinic acid (ALA) synthetase and contents of cytochrome P-450 and the microsomal haem were decreased in the liver. In contrast, in the kidney, cytochrome P-450 and haem concentrations were significantly increased, with no change in ALA synthetase activity. The increase in the kidney haem content appeared to reflect an increased formation of haem, as suggested by the elevated activity of ferrochelatase and the concomitant decrease in tissue porphyrin levels. In the kidney, a time-dependent and pronounced inhibition of activities of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione production, and gamma-glutamyl transpeptidase, the first enzyme in glutathione breakdown, were observed. The enzyme activities, 7 days after treatment, were only 40 and 60% of the control values respectively. In contrast, these enzyme activities were not affected in the liver. Complexing cis-platinum with cysteine considerably intensified the entire spectrum of effects of cis-platinum in the kidney. Notably, cytochrome P-450 concentration and haem oxygenase activity were increased to about 3.5 and 6 times the control values, respectively. gamma-Glutamylcysteine synthetase activity was decreased to less than 20% of the control. It is suggested that the differential effectiveness of cis-platinum in the liver and the kidney in alternating haem metabolism is related to the vast differences which exist between these organs in the activities of gamma-glutamyl-cycle enzymes. It is further suggested that this may promote the formation in the kidney, but not in the liver, of a cis-platinum-cysteine complex that is more stable, and thus biologically more effective, than the parent compound.

Project description:Glutathione is a tripeptide composed of glutamate, cysteine and glycine. Glutathione is present in millimolar concentrations in most mammalian cells and it is involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission/neuromodulation. Glutathione is metabolised via the gamma-glutamyl cycle, which is catalyzed by six enzymes. In man, hereditary deficiencies have been found in five of the six enzymes. Glutathione synthetase deficiency is the most frequently recognized disorder and, in its severe form, it is associated with hemolytic anemia, metabolic acidosis, 5-oxoprolinuria, central nervous system (CNS) damage and recurrent bacterial infections. Gamma-glutamylcysteine synthetase deficiency is also associated with hemolytic anemia, and some patients with this disorder show defects of neuromuscular function and generalized aminoaciduria. Gamma-glutamyl transpeptidase deficiency has been found in patients with CNS involvement and glutathionuria. 5-Oxoprolinase deficiency is associated with 5-oxoprolinuria but without a clear association with other symptoms. Dipeptidase deficiency has been described in one patient. All disorders are very rare and inherited in an autosomal recessive manner. Most of the mutations are leaky so that many patients have residual enzyme activity. Diagnosis is made by measuring the concentration of different metabolites in the gamma-glutamyl cycle, enzyme activity and in glutathione synthetase and gamma-glutamylcysteine synthetase deficiency, also by mutation analysis. Prenatal diagnosis has been preformed in glutathione synthetase deficiency. The prognosis is difficult to predict, as few patients are known, but seems to vary significantly between different patients. The aims of the treatment of glutathione synthesis defects are to avoid hemolytic crises and to increase the defense against reactive oxygen species. No treatment has been recommended for gamma-glutamyl transpeptidase, 5-oxoprolinase and dipeptidase deficiency.

Dataset Information

Assay, purification, properties and mechanism of action of gamma-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure