Inducers of gamma-glutamylcysteine synthetase and their effects on glutathione synthetase expression

Synthesis of GSH occurs via two enzymatic steps, the first is catalyzed by gamma-glutamylcysteine synthetase (GCS) and the second is catalyzed by GSH synthetase (GS). A heavy (HS) and light subunit (LS) make up GCS; regulation of both subunits have been well characterized, whereas regulation of GS is largely unknown. In this study, we examined the effects of treatments known to influence the gene expression of GCS subunits on GS expression. Insulin and hydrocortisone treatment of rat hepatocytes or ethanol-feeding of rats for 9 weeks, which increased the expression of GCS-HS only, had no influence on GS expression. However, two-thirds partial hepatectomy in rats which increased the expression of GCS-HS only, also increased GS expression. Treatment of hepatocytes or rats with diethyl maleate, buthionine sulfoximine, tert-butylhydroquinone, or thioacetamide, which increased the expression of both GCS subunits, increased the expression of GS. The GSH synthesis capacity increased 50-100% by treatments that increased only the GCS-HS expression, whereas it increased 161-200% by treatments that increased both GCS-HS and GS expression. Thioacetamide treatment of Chang cells increased cell GSH and GS expression by 50%, but had minimal influence on GCS subunits. Thus, GS induction can further increase the cell's GSH synthetic capacity and in some cells may be as important as GCS in determining the rate of GSH synthesis.     

Bovine lens gamma-glutamylcysteine synthetase. Inhibition by glutathione and adenine nucleotides

Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme . L-glutamate forms of gamma-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5'-diphosphate (5'ADP) is also an inhibitor and competes with both ATP and L-beta-chloroalanine for Mg . enzyme . L-glutamate and Mg . enzyme . L-glutamylphosphate, respectively. Under physiological conditions in the lens, 5' ADP competes effectively with L-cysteine for Mg . enzyme . L-glutamylphosphate, owing to the low concentration of L-cysteine, and less effectively with ATP for Mg . enzyme . L-glutamate, because of a high concentration of ATP.     

Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

Glutathione (GSH) is synthesized by gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed gamma-GCS-GS catalyzing both gamma-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the gamma-GCS activity, S. agalactiae gamma-GCS-GS had different substrate specificities from those of Escherichia coli gamma-GCS. Furthermore, S. agalactiae gamma-GCS-GS synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-X(aa)-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding gamma-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae gamma-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed gamma-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-Cys-X(aa). Whereas the substrate specificities of gamma-GCS domain protein and GS domain protein of S. agalactiae gamma-GCS-GS were the same as those of S. agalactiae gamma-GCS-GS.     

Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity.

Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the catalytic subunit of GCS. These mutant cells contained a wild-type steady-state level of GSH and, after depletion, synthesized GSH at the same rate as wild-type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in mutant cells by 80% but did not increase that of wild-type cells, and, in GSH-depleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard.     

A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells

An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.     

Disruption of gamma-glutamylcysteine synthetase results in absolute glutathione auxotrophy and apoptosis in Candida albicans

Glutathione is the most abundant non-protein thiol and a major source of reducing equivalents in eukaryotes. We examined the role of glutathione in Candida albicans by the disruption of gamma-glutamylcysteine synthetase (GCS1), an essential enzyme in glutathione biosynthesis. The gcs1/gcs1 null mutants exhibited glutathione auxotrophy, which could be rescued by supplementing with reduced and oxidized glutathione and gamma-glutamylcysteine. When the mutants were depleted of glutathione, they showed typical markers of apoptosis. These results suggest that glutathione itself is an essential metabolite and C. albicans lacking GCS1 undergoes apoptosis.     

Glutathione metabolism in primate lenses: A phylogenetic study of glutathione synthesis and glutathione redox cycle enzyme activities

Lens wet weights, soluble protein, and activities of γ-glutiamylcysteine synthetase, glutathione synthetase, glutathione peroxidase, and glutathione reductase were determined in primate lenses. The primary sources of lenses were middle-aged adult animals. The Primates, from 23 genera, were categorized into six superfamilies: hominoids (five species), Old World monkeys (seven species), New World monkeys (five species), tarsiers (two species), lemurs (six species), and lorisids (three species). Significant differences between various groups or combinations of groups were noted for γ-glutamylcysteine synthetase, glutathione peroxidase, and glutathione reductase activities. Lenticular γ-glutamylcysteine synthetase activity was very low in the Old World simian lenses and highest in the prosimians. Glutathione peroxidase activity was extraordinarily high in lenses of Old World monkeys. Glutathione reductase activity was low in all the prosimians but tenfold higher in hominoid lenses with intermediate values in monkeys of both the Old World and New World. Glutathione synthetase activity was variable, and no clear pattern which might be useful for primate classification was noted. Lenticular activity ratios of glutathione synthetase:γ-glutamylcysteine synthetase were highest in the Old World simians and lowest in the prosimians. These data with emphasis upon Aotus and the tarsiers were examined with regard to phylogenetic relationships. © 1994 Wiley-Liss, Inc.     

Glutathione peroxidases and glutathione reductase activities during Bufo bufo development

Glutathione peroxidases and glutathione reductase activities are expressed from the early stage of Bufo bufo development. Selenium-dependent and selenium-independent glutathione peroxidase activities fluctuated independently. The activity of selenium-independent was found to be higher than that of selenium-dependent glutathione peroxidase through all stages of development. Glutathione reductase activity, after a slight fall from stage 4 to stage 7, constantly increased up to stage 25.     

Vaccination with recombinant Leishmania donovani gamma-glutamylcysteine synthetase fusion protein protects against L. donovani infection

Visceral leishmaniasis presents a serious health threat in many parts of the world. There is, therefore, an urgent need for an approved vaccine for clinical use to protect against infection. In this study, the ability of recombinant Leishmania donovani gamma-glutamyl cysteine synthetase protein (LdγGCS) alone or incorporated into a non-ionic surfactant vesicle (NIV) delivery system to protect against L. donovani infection was evaluated in a BALB/c mouse model. Immunization with LdγGCS alone or LdγGCS-NIV induced specific IgG1 and IgG2a antibodies compared to controls, with LdγGCS-NIV inducing significantly higher titers of both antibody classes (P < 0.05). Both formulations induced similar increases in splenocyte IFN-γ production following ex vivo antigen stimulation with LdγGCS compared with cells from control mice (P < 0.05). Similar levels of protection against infection were induced by LdγGCS alone and LdγGCS-NIV, based on their ability to suppress liver parasite burdens compared to control values (P < 0.01), indicating that using a carrier system did not enhance the protective responses induced by the recombinant protein. The results of this study indicate that LdγGCS may be a useful component in a vaccine against L. donovani.     

Branched-chain amino acid transport in Streptococcus agalactiae.

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.     

Regulation of gamma-glutamylcysteine synthetase expression in response to oxidative stress.

Glutathione (GSH) is synthesized by the activity of two ATP-requiring GSH synthesizing enzymes. Gamma-glutamylcysteine synthetase (gamma-GCS) is the rate limiting enzyme for the GSH synthesis. Gamma-GCS is a heterodimer of heavy, catalytic subunit and light, regulatory subunit and responsive to many stresses, such as heat shock, oxidative stress or cytokines. To know the regulation of the expression of gamma-GCS gene, in the present study, we show evidences that gamma-GCS heavy subunit is upregulated by oxidative stress by ionizing radiation and TNF-alpha mediated by nuclear factor-kappaB (NF-kappaB), and impairment of the expression of gamma-GCS by TNF-alpha in diabetic condition. Furthermore we describe the importance of GSH in the regulation of NF-kappaB subunits.     

Enhancement of gamma-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury.

Glutathione (GSH), a major cellular antioxidant, is elevated 2- to 3-fold in kidneys of rats during prolonged treatment with mercury as methyl mercury hydroxide (MMH). Increased renal GSH is accompanied by a dose- and time-related elevation in the relative abundance of mRNA hybridizable to a cDNA probe which encodes renal gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Renal GCS mRNA is maximally elevated 4.4-fold at 3 weeks following initiation of MMH treatment. Enhancement of GSH and GCS mRNA content corresponds to a relative sparing of renal cells from oxidative tissue damage during MMH exposure. These observations suggest that increased synthesis of GSH at the genetic level occurs as an initial adaptive response to mercury-induced oxidative stress in kidney cells.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.