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.
Project description:Spectrophotometric assay methods are described for glutathione synthetase, gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase of erythrocytes. The contents of these enzymes in normal human erythrocytes are reported. Erythrocyte glutathione synthetase is inhibited by ADP; this inhibition is competitive with respect to ATP. gamma-Glutamylcysteine synthetase is subject to feedback inhibition by GSH, and is also inhibited by NADH, and to a lesser extent by NAD(+) and NADPH. This enzyme is irreversibly inactivated by cysteamine.
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:GSH is the major low-molecular-mass thiol in most organisms. The tripeptide maintains a reduced intracellular environment and protects cellular components from damaging oxidation. GSH is synthesized by the action of two ATP-dependent enzymic steps, in which gamma-glutamylcysteine synthetase (gamma-GCS) catalyses the ligation of glutamate and cysteine and subsequently glutathione synthetase (GS) adds glycine to the dipeptide. Recently it was shown that the synthesis of gamma-glutamylcysteine is crucial for the survival of the erythrocytic stages of the malaria parasite Plasmodium falciparum by using the specific gamma-GCS inhibitor buthionine sulphoximine. In order to investigate further the synthetic pathway of the tripeptide in the parasite, GS was cloned and expressed recombinantly. The deduced amino acid sequence of P. falciparum GS shares only a moderate degree of identity with other known GSs, but the residues responsible for substrate and co-factor binding are almost all conserved, with the exception of the ones involved in gamma-glutamylcysteine binding. The protein is active as a dimer, with a subunit molecular mass of 77 kDa, and the addition of reducing reagents such as dithiothreitol is essential in maintaining enzymic activity, indicating that thiol groups are important for stability and enzymic activity. The K(app)(m) values for gamma-glutamyl-alpha-aminobutyrate, ATP and glycine were determined to be 107.1 microM, 59.1 microM and 5.04 mM, respectively, and the V(max) of 5.24 +/- 0.7 micromol.min(-1).mg(-1) was in the same range as that of the mammalian enzymes. However, the negative co-operativity observed for gamma-glutamylcysteine binding to the rat enzyme was not found for the parasite protein. This may be due to the alteration of several amino acids in the gamma-glutamylcysteine-binding site.
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.
Project description:A Na(+)-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na(+)-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na(+)-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na(+)-dependent taurocholate uptake, but it blocked Na(+)-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na(+)-dependent and Na(+)-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver.
Project description:Chinese pickled cabbage is a traditional fermented food that contains abundant microbes produced during the process of fermentation. In this work, an in vivo animal study was conducted to investigate the effects of a newly isolated lactic acid bacterium (Lactobacillus plantarum CQPC11, LP-CQPC11) on d-galactose-induced oxidation and aging in mice. Analysis of the serum and tissue samples of these mice using molecular biology approaches showed that LP-CQPC11 suppressed the decrease in thymus, brain, heart, liver, spleen, and kidney indices caused by oxidation and aging. Furthermore, LP-CQPC11 increased the levels of SOD (superoxide dismutase), GSH-Px (glutathione peroxidase), and GSH (glutathione), whereas it reduced the levels of NO (nitric oxide) and MDA (malondialdehyde) in the serum, liver, and spleen of oxidation and aging mouse models. Pathological observation indicated that LP-CQPC11 alleviated the damage caused by oxidation and aging on the liver and spleen of mice. qPCR analysis indicated that LP-CQPC11 effectively upregulated the expression of nNOS (neuronal nitric oxide synthase), eNOS (endothelial nitric oxide synthase), Cu/Zn-SOD (cuprozinc-superoxide dismutase), Mn-SOD (manganese superoxide dismutase), CAT (catalase), HO-1 (heme oxygenase-1), Nrf2 (nuclear factor-erythroid 2 related factor 2), γ-GCS (γ-glutamylcysteine synthetase), and NQO1 (NAD(P)H dehydrogenase [quinone] 1), but downregulated the expression of iNOS (inducible nitric oxide synthase) in the mouse liver and spleen. Western blot analysis showed that LP-CQPC11 effectively upregulated SOD1 (Cu/Zn-SOD), SOD2 (Mn-SOD), CAT, GSH1 (c-glutamylcysteine synthetase), and GSH2 (glutathione synthetase) protein expression in mouse liver and spleen tissues. These findings suggest that LP-CQPC11 can effectively prevent d-galactose-induced oxidation and aging in mice, and the effect is even better than that of the commonly used Lactobacillus delbruechii subsp. bulgaricus (LDSB) and vitamin C in the industry. Thus, LP-CQPC11 may be potentially employed as a probiotic strain.
Project description:A mutant of Escherichia coli, JTG10, deficient in gamma-glutamylcysteine synthetase (gamma-ECS; EC 18.104.22.168) is unable to synthesize glutathione (GSH) and is sensitive to 8-hydroxyquinoline. This phenotype was exploited for the isolation of Arabidopsis thaliana gamma-ECS cDNAs by expression cloning, and clones were selected through functional complementation by growth on 8-hydroxyquinoline. High levels of gamma-ECS activity were detectable in extracts derived from cultures of JTG10 expressing the Arabidopsis gamma-ECS open reading frame, although these complemented mutants accumulated GSH to only 10% of the wild-type level. The derived amino acid sequence constitutes a polypeptide of 59.9 kDa and shows only 44-48% similarity with previously published sequences of rat kidney, human liver, yeast, and E. coli gamma-ECS. When the gamma-ECS cDNA was used as a probe, Southern blot analysis of Arabidopsis genomic DNA revealed that it is present as a low copy number gene. Furthermore, the Arabidopsis gamma-ECS cDNA probe failed to hybridize to maize and tobacco genomic DNA at low stringency, suggesting that heterogeneity in gamma-ECS structure exists between plant species. The activity of recombinant Arabidopsis gamma-ECS was inhibited by buthionine sulfoximine and GSH, indicating that, while differences in the primary and secondary structure of gamma-ECS from different sources exist, the enzymes may have similar active site structures.
Project description:Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol-MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC.
Project description:The formation of gamma-glutamylhydroxamate by homogenates under optimum assay condition showed an inconstancy in the ratios of the enzyme activities utilizing l-glutamate and ATP (gamma-glutamylhydroxamate synthetase) and l-glutamine and ADP (l-glutamine-hydroxylamine glutamyltransferase) in a number of normal and neoplastic rat tissues. Although gamma-glutamylhydroxamate synthetase activities in adult livers and kidneys were identical in males and females, l-glutamine-hydroxylamine glutamyltransferase activities in the organs of females were significantly lower. The developmental formations of the two activities in liver, kidney, brain and muscle were not simultaneous. The l-glutamine-hydroxylamine glutamyltransferase activity in foetal liver or neonatal kidney could be prematurely evoked by thyroxine, but the gamma-glutamylhydroxamate synthetase activity remained unchanged. Injections of cortisol also had dissimilar effects on the two activities in thymus and hepatomas. The discrepant tissue distribution, asynchronous developmental formation and differential response to several hormonal stimuli provide evidence in vivo that the two activities are not catalysed by the same protein.
Project description:By a study of the product-inhibition kinetics of the octanoyl-CoA synthetase from ox liver mitochondria, evidence was obtained consistent with the hypothesis that the enzyme reacts by a Bi Uni Uni Bi Ping Pong type of mechanism in which the order of addition and evolution of substrates and products is CoA, octanoate, octanoyl-CoA, ATP, PP(i) and AMP. There is also evidence that more than one molecule of CoA can add to the enzyme and that it may act as an allosteric activator.