Glutathione Synthetase Activity Research Articles

A Comparative Analysis of Chemical Constituents and Antioxidant Effects of Dendrobium fimbriatum Hook Fractions with Different Polarities.

The aim of this study was to investigate the chemical composition and antioxidant capacity of various polar fractions obtained from Dendrobium fimbriatum Hook (DH). First, a 90% ethanol-aqueous extract of DH (CF) was subjected to sequential fractionation using different organic solvents, resulting in the isolation of a methylene chloride fraction (DF), an ethyl acetate fraction (EF), an n-butanol fraction (BF), and a remaining water fraction (WF) after condensation. Additionally, the CF was also subjected to column chromatography via a D101 macroreticular resin column, eluted with ethanol-aqueous solution to yield six fractions (0%, 20%, 40%, 60%, 80%, and 100%). UPLC-Q-Exactive Orbitrap-MS/MS analysis identified a total of 47 chemical compounds from these polar fractions, including fatty acids, amino acids, phenolic acids, flavonoids, organic heterocyclic molecules, and aromatic compounds. Moreover, DF, EF, and the 60%, 80%, and 100% ethanol-aqueous fractions had higher total phenol content (TPC) and total flavonoid content (TFC) values and greater 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS-) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging abilities. In H2O2-induced HepG2 cells, the aforementioned fractions could increase the activities of antioxidative enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), heme oxygenase-1 (HO-1) and catalase (CAT), stimulate glutathione (GSH) synthesis by increasing the activities of glutamic acid cysteine ligase (GCL) and glutathione synthetase (GS), regulate GSH metabolism by increasing glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activities, and reduce levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Furthermore, the antioxidative stress effect of the DH fractions was found to be positively correlated with the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) protein and the presence of antioxidative chemical constituents. In conclusion, this study highlights the efficacy of both liquid-liquid extraction and macroporous resin purification techniques in the enrichment of bioactive compounds from natural food resources. The comprehensive analysis of chemical constituents and antioxidant effects of different polar fractions from Dendrobium fimbriatum Hook contributes to the understanding of its potential application in functional foods and nutraceuticals.

Exploring Idiopathic Pulmonary Fibrosis Biomarker by Simultaneous Two-Photon Fluorescence Imaging of Cysteine and Peroxynitrite.

Idiopathic pulmonary fibrosis (IPF) has been characterized as a chronic inflammatory disease that leads to irreversible damage to pulmonary function. However, there is no specific IPF biomarker that can be used to distinguish IPF and not pneumonia. Endoplasmic reticulum (ER) stress is prominent in IPF. To search for a specific biomarker of IPF, we developed two ER-targeting two-photon (TP) fluorescent probes, TPER-ONOO and TPER-Cys, for peroxynitrite (ONOO-) and cysteine (Cys) imaging, respectively. A significant increase in Cys levels in the lungs was discovered only in mice with IPF, which implied that Cys might be an IPF biomarker candidate. Furthermore, we uncovered the mechanism of glutathione (GSH) deficiency in IPF, which was not due to Cys shortage but instead was attributable to impaired glutamate cysteine ligase and glutathione synthetase activities via ONOO--induced post-transcriptional modification. This work has potential to provide a new method for IPF early diagnosis and drug efficacy evaluation.

Effect of ozone treatment on glutathione (GSH) status in selected berry fruit

The major aim of this study was to investigate the effect of one-time ozone elicitation on glutathione metabolism in selected berry fruit. Raspberry, highbush blueberry, strawberry, blackberry and blackcurrant fruit was ozonated with an ozone concentration of 15 ppm for 30 min. Research showed that ozonation process causes clear and positive changes in the metabolism of glutathione in berries. After ozonation, the fruit was characterized by increased level of glutathione (GSH), which resulted from higher activity of glutathione synthetase. Moreover, ozonation improved the metabolism activity of mitochondria leading to an increase in biosynthesis of ATP, which is the source of energy necessary for the GSH production. The higher activity of the enzymes involved in glutathione metabolism i.e. glutathione peroxidase and glutathione reductase and increased level of glutathione contributed to the higher ability of the fruit to scavenging the hydrogen peroxide.

Arginine glycosylation enhances methylglyoxal detoxification

Type III secretion system effector proteins have primarily been characterized for their interactions with host cell proteins and their ability to disrupt host signaling pathways. We are testing the hypothesis that some effectors are active within the bacterium, where they modulate bacterial signal transduction and physiology. We previously determined that the Citrobacter rodentium effector NleB possesses an intra-bacterial glycosyltransferase activity that increases glutathione synthetase activity to protect the bacterium from oxidative stress. Here we investigated the potential intra-bacterial activities of NleB orthologs in Salmonella enterica and found that SseK1 and SseK3 mediate resistance to methylglyoxal. SseK1 glycosylates specific arginine residues on four proteins involved in methylglyoxal detoxification, namely GloA (R9), GloB (R190), GloC (R160), and YajL (R149). SseK1-mediated Arg-glycosylation of these four proteins significantly enhances their catalytic activity, thus providing another important example of the intra-bacterial activities of type three secretion system effector proteins. These data are also the first demonstration that a Salmonella T3SS effector is active within the bacterium.

Mitigation of arsenate toxicity by indole-3-acetic acid in brinjal roots: Plausible association with endogenous hydrogen peroxide

The role of indole-3-acetic acid (IAA) and hydrogen peroxide (H2O2) crosstalk in regulating metal stress is still less known. Herein, role of IAA in alleviating arsenate (AsV) toxicity in brinjal seedlings along with its probable relation with endogenous H2O2 was investigated. Arsenate hampered root growth due to greater accumulation of As and decrease in phosphorus uptake that resulted into inhibited photosynthesis and cell death. Further, AsV induced oxidative stress markers and damage to macromolecules (lipids and proteins) due to alterations in redox status of glutathione as a result of inhibition in activity of glutathione synthetase and glutathione reductase. However, application of IAA with AsV improved root growth by significantly declining As accumulation and oxidative stress markers, sequestrating As into vacuoles, and improving redox status of glutathione which collectively protected roots from cell death. Interestingly, addition of diphenylene iodonium (DPI, an inhibitor of NADPH oxidase) further increased AsV toxicity even in the presence of IAA. However, application of H2O2 rescued negative effect of DPI. Overall, the results suggested that in IAA-mediated mitigation of AsV toxicity in brinjal roots, endogenous H2O2 might have acted as a downstream signal.

Kinetics of inhibition of isoproturon to glutathione-associated enzymes in wheat.

The present study aimed at investigating the kinetic of inhibition of isoproturon to the GSH-associated enzymes [γ-glutamyl-cysteine synthetase (γ-GCS), glutathione synthetase (GS), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione peroxidase (GPX)] in wheat. Isoproturon, applied to 10-day-old seedlings for the following 12days, provoked significant reductions in shoot fresh and dry weights, protein, thiols and glutathione (GSH); however, oxidized glutathione (GSSG) was elevated while GSH/GSSG ratio was declined with concomitant significant inhibitions in the activities of γ-GCS, GS, GR, GST and GPX; the effect was time dependent. IC50 and Ki values of isoproturon were lowest for GPX, highest for both GST and GR, and moderate for both γ-GCS and GS. The herbicide markedly decreased Vmax of γ-GCS, GS and GPX but unchanged that of GST and GR; however, Km of γ-GCS, GS, GST and GR increased but unchanged for GPX. The pattern of response of changing Vmax, Km, Vmax/Km, kcat and kcat/Km for in vivo and in vitro tests of each enzyme seemed most likely similar. These results indicate that a malfunction to defense system was induced in wheat by isoproturon resulting in inhibitions in GSH-associated enzymes, the magnitude of inhibition was most pronounced in GPX followed by γ-GCS, GS, GST, and GR. These findings could conclude that isoproturon competitively inhibited GST and GR; however, the inhibition was noncompetitive for GPX but mixed for both γ-GCS and GS.

Changes of lipid peroxidation and glutathione redox system, and expression of glutathione peroxidase regulatory genes as effect of short-term aflatoxin B1 exposure in common carp

Lipid peroxidation, glutathione content, glutathione peroxidase activity and gene expression of transcription factors, glutathione peroxidase, glutathione synthetase and glutathione reductase was investigated in common carp liver. Short term (24 h) exposure of aflatoxin B1 at different doses (100, 200 and 400 μg AFB1 kg−1 feed) was used. It was found that conjugated dienes and trienes elevated after 16 h, while thiobarbituric acid reactive substances content increased only at the lowest dose after 16 and 24 h of exposure. Glutathione content showed higher levels than control after 16 h of exposure and glutathione peroxidase (GPx4) activity was higher in all of the AFB1 treated than the control group after 8 h of exposure. Gene expression of transcription factors showed dual response. Expression of keap1 gene down-regulated after 8 h and 16 h and nrf2 gene after16 h, but up-regulated after 24 h of exposure in the lowest, and highest dose groups. Expression of gpx4a and gpx4b genes down-regulated after 8 h and induction was found after 16 and 24 h of exposure, irrespective of the dose. The results indicated that low dose of AFB1 provokes oxidative stress earlier than higher doses, which activated the Keap1-Nrf2 pathway. At higher doses this pathway activated later, but preformed GPx4 effectively prevented lipid peroxidation.

Exogenous 24-epibrassinolide alleviates zinc-induced toxicity in eggplant (Solanum melongena L.) seedlings by regulating the glutathione-ascorbate-dependent detoxification pathway

ABSTRACTExcessive zinc (Zn) uptake is toxic to both plants and animals. 24-Epibrassinolide (EBL), a plant steroid hormone, plays a pivotal role in regulating plant resistance to various stresses. In this study, the effects of EBL on oxidative stress and components of the ascorbate–glutathione (AsA-GSH) cycle in the leaves of eggplant (Solanum melongena L.) seedlings were investigated under Zn stress. Exogenous 0.1 µM EBL significantly alleviated the oxidative damage caused by Zn stress, as reflected by increased plant growth and decreased accumulations of Zn, hydrogen peroxide (H2O2), and malondialdehyde (MDA). In addition, components of the AsA-GSH metabolism cycle, such as the concentrations of reduced ascorbic acid (AsA) and glutathione (GSH), as well as the activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), and glutathione synthetase (GS), increased significantly in the presence of EBL under Zn stress. Moreover, exogenous EBL caused a significant increase in the levels of expression of the DHAR, MDHAR, GR, and GS genes in the AsA-GSH cycle during Zn toxicity, showing that EBL could regulate the regeneration of AsA and GSH at the transcriptional level. Taken together, the results of the present study suggest that exogenous EBL confers resistance against Zn-induced oxidative damage by enhancing the ASA-GSH cycle by up-regulating key gene expression and key enzyme activities.

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

1. Metabolic acidosis due to accumulation of l-5-oxoproline is a rare, poorly understood, disorder associated with acetaminophen treatment in malnourished patients with chronic morbidity. l-5-Oxoprolinuria signals abnormal functioning of the γ-glutamyl cycle, which recycles and synthesises glutathione. Inhibition of glutathione synthetase (GS) by N-acetyl-p-benzoquinone imine (NAPQI) could contribute to 5-oxoprolinuric acidosis in such patients. We investigated the interaction of NAPQI with GS in vitro.2. Peptide mapping of co-incubated NAPQI and GS using mass spectrometry demonstrated binding of NAPQI with cysteine-422 of GS, which is known to be essential for GS activity. Computational docking shows that NAPQI is properly positioned for covalent bonding with cysteine-422 via Michael addition and hence supports adduct formation.3. Co-incubation of 0.77 μM of GS with NAPQI (25–400 μM) decreased enzyme activity by 16–89%. Inhibition correlated strongly with the concentration of NAPQI and was irreversible.4. NAPQI binds covalently to GS causing irreversible enzyme inhibition in vitro. This is an important novel biochemical observation. It is the first indication that NAPQI may inhibit glutathione synthesis, which is pivotal in NAPQI detoxification. Further studies are required to investigate its biological significance and its role in 5-oxoprolinuric acidosis.

Hemolytic Anemia and Metabolic Acidosis: Think about Glutathione Synthetase Deficiency

Glutathione synthetase deficiency (GSSD) is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with hemolytic anemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low glutathione synthetase activity in erythrocytes or cultured skin fibroblasts. The prognosis seems to depend on early diagnosis and treatment. We report a 4 months old Tunisian male infant who presented with severe metabolic acidosis with high anion gap and hemolytic anemia. High level of 5-oxoproline was detected in her urine and diagnosis of GSSD was made. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. He died of severe metabolic acidosis and sepsis at the age of 15 months.

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn2+) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn2+ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn2+ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn2+ induced oxidative stress.

Acute exposure to 3-methylcholanthrene induces hepatic oxidative stress via activation of the Nrf2/ARE signaling pathway in mice

Polycyclic aromatic hydrocarbons (PAHs) are the most common contaminants in the environment. The primary focus on the toxicity of PAHs is their ability to activate the aryl hydrocarbon receptor (AhR)-mediated pathway and lead to carcinogenesis in different organisms. However, the influence of PAHs on the antioxidant system in mammalian systems has received only limited attention. In the present study, we observed that the intraperitoneal injection of 100 mg/kg 3-methylcholanthrene (3MC) into mice significantly increased reactive oxygen species (ROS) levels and malondialdehyde (MDA) contents and decreased glutathione (GSH) contents and the activity of total antioxidant capacity (T-AOC), indicating that serious oxidative stress had been induced in the liver of mice. Then, the oxidative stress signal activated the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway by enhancing the mRNA levels of Nrf2, p38, and Erk2. Moreover, the mRNA levels of Nrf2/ARE target genes, including glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione synthetase (GS), NAD(P)H: quinone oxidoreductase 1 (Nqo1), superoxide dismutase 1 (Sod1), and Sod2, increased significantly after treatment with 3MC for 24 hours. The hepatic levels of NQO1 and the activities of GR and GS were also significantly enhanced at 24 hours after 3MC treatment. Because the expression of NQO1 is co-regulated by Nrf2/ARE and AhR/XRE in mammalian tissues, NQO1 may play an important role in protecting against the oxidative stress induced by 3MC. Taken together, our findings suggested that acute exposure to 3MC altered the cellular redox balance in hepatocytes to trigger Nrf2-regulated antioxidant responses, which may represent an adaptive cell defense mechanism against the oxidative stress induced by PAHs.

Thiol-based redox signaling in the nitrogen-fixing symbiosis

In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N 2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS) so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed us to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, that their spatio-temporally distribution is correlated with the corresponding (homo)glutathione synthetase activities, and that they are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume-rhizobial symbiosis.

Glutathione redox cycle dysregulation in Huntington’s disease knock-in striatal cells

Huntington’s disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and γ-glutamyl transpeptidase (γ–GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to counterbalance increased ROS formation and emergent apoptotic features in HD striatal cells.

High concentration of cadmium induces AtPCS2 gene expression in Arabidopsis thaliana (L.) Heynh ecotype Wassilewskija seedlings

In Arabidopsis thaliana, two genes encoding phytochelatin synthase (PCS; EC 2.3.2.15), AtPCS1 and AtPCS2, have been identified. Until now, only AtPCS1 was shown to play a role in response to Cd. To gain insight into the putative role of AtPCS2, three Cd concentrations (50, 100 and 200 μM) and long-term exposure (7 days) were tested on 1-week-old A. thaliana ecotype Wassilewskija (Ws) seedlings. Since 100 μM Cd did not alter seedling metabolism, as shown by unchanged total soluble protein and free proline contents, we investigated plantlet response to this concentration in addition to Cd accumulation. Seedlings accumulated Cd in roots and shoots. As phytochelatins and glutathione (GSH) contents increased in treated seedlings, we suggested that Cd might be translocated via the phytochelatin pathway. Specific enzymatic activities of γ-glutamylcysteine synthetase (GCS; EC 6.3.2.2), glutathione synthetase (GS; EC 6.3.2.3) and PCS were twice much more stimulated in shoots and roots after Cd exposure except GS that remained constant in shoots. As expression of genes encoding GCS and GS was unchanged in response to Cd, we suggested a regulation at translational or post-translational level. Surprisingly, AtPCS1 and AtPCS2 were differentially up-regulated after Cd treatment: AtPCS1 in shoots and AtPCS2 in whole plantlets. This last result suggests that PCS2 could be involved in plant response to high concentration of Cd in Ws ecotype and supports a putative role of PCS2, not redundant with PCS1, in a long-term response to Cd.

Glutathione is essential for early embryogenesis – Analysis of a glutathione synthetase knockout mouse

Glutathione (GSH) is present in all mammalian tissues and plays a crucial role in many cellular processes. The second and final step in the synthesis involves the formation of GSH from gamma-glutamylcysteine (γ-GC) and glycine and is catalyzed by glutathione synthetase (GS). GS deficiency is a rare autosomal recessive disorder, and is present in patients with a range of phenotypes, from mild hemolytic anemia and metabolic acidosis to severe neurologic disorders or even death in infancy. The substrate for GS, γ-GC, has been suggested as playing a protective role, by substituting for GSH as an antioxidant in GS deficient patients. To examine the role of GS and GSH metabolites in development, we generated mice deficient in GSH by targeted disruption of the GS gene ( Gss). Homozygous mice died before embryonic day (E) 7.5, but heterozygous mice survived with no distinct phenotype. GS protein levels and enzyme activity, as well as GSH metabolites, were investigated in multiple tissues. Protein levels and enzyme activity of GS in heterozygous mice were diminished by 50%, while GSH levels remained intact. γ-GC could not be detected in any investigated tissue. These data demonstrate that GSH is essential for mammalian development, and GSH synthesis via GS is an indispensable pathway for survival.

Production of glutathione by using Yarrowia lipolytica enhanced the activity of glutathione synthetase

Production of glutathione by using Yarrowia lipolytica enhanced the activity of glutathione synthetase

HIF1 Induced Glutathione Dysregulation in Platelets Associated with Increased Risk of Vascular Disease in Chuvash Polycythemia.

The von Hippel-Lindau protein-hypoxia-inducible factor pathway is a transcriptional system controlling cellular responses to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of α and β subunits. Under normoxia, the prolyl residues of the α subunit are hydroxylated allowing the von Hippel-Lindau protein (pVHL) to bind, which targets HIFα for proteasomal degradation. During hypoxia, HIFα does not bind to pVHL and instead forms a transcriptional complex with HIFβ leading to increased expression of a broad range of hypoxia-regulated genes. Patients with Chuvash polycythemia (CP) are homozygous for a 598 C->T mutation in the VHL gene, resulting in a pVHL that causes ineffective degradation of HIFα. Clinically, CP patients suffer from premature mortality related to vascular thrombotic events - an increased risk that is not related to their elevated hematocrit, blood pressure, or known cardio or cerebrovascular risk factors. Lab abnormalities seen in CP include erythrocytosis and an elevated plasma glutathione level (Sergueeva et al, Haematologica. Feb 2008). Glutathione (GSH) plays an essential role in cellular antioxidant protection, and its levels are controlled by two mechanisms. GSH is oxidized to glutathione disulfide (GSSG) but is replenished by reduction of GSSG via glutathione reductase (GSR). GSH is also increased by de novo synthesis, which is regulated at several levels, including GSH feedback inhibition, and glutathione synthetase (GSS) and glutamate cysteine ligase (GCL) activity. To establish the molecular basis of elevated GSH, we examined expression of GSS, GSR, and GCL in the platelets of 11 CP patients and 8 Chuvash controls using qRT-PCR. Analysis revealed a 2-fold increased expression of GCL in CP. GSR and GSS were not statistically different. This data suggests that increased GCL activity might be the mechanism by which GSH is elevated in CP, but whether HIF directly regulates GCL or whether these differences reflect a more global process are presently unknown. Since the promoter of GCL contains an oxidative stressresponse element, transcriptional up regulation of GCL by increased oxidative stress secondary to HIF dysregulation might drive increased GSH synthesis. To examine this question, we measured GSSG in our samples. Although CP patients had elevated GSH (8.00 uM vs. 4.32 uM, p 0.002), the GSH/GSSG ratio (a widely used marker of redox state) showed no differences between CP and controls. Thus, overexpression of GCL and elevated GSH appear not to be compensatory responses to increased oxidative stress in CP. We also found increased GCL expression in VHL mutant mice. To determine if HIF1 might regulate this expression, we next measured GCL expression in HIF1 deficient embryos at embryonic day 9.5 and found decreased expression of GCL. In the homozygote knock-out embryos where HIF1 is absent, GCL expression is decreased, in contrast to CP patients, where HIF1 and GCL expression are both increased. Collectively, our data suggests that HIF1 dysregulates cellular redox homeostasis by upregulating GCL and increasing GSH synthesis in an oxidative-stress-independent manner. The significance of elevated GSH in CP and its possible relationship to increased thromboses remains to be defined. Reactive oxygen species mediate the vascular inflammation seen in the development of atherosclerotic disease, and GSH is an important intracellular scavenger that protects cells against oxidative damage. Yet CP patients have increased GSH as well as increased thrombosis, which is especially provoking since recently published evidence suggests that increased redox potential may be harmful to the cardiovascular system (Rajasekaran, et al Cell 2007). Perhaps the GSH elevation may contribute to the increased vascular disease that constitutes the major cause of mortality in this disorder of hypoxia sensing. The cellular antioxidant defense system is intimately linked to oxidative stress, hypoxia regulation, and vascular homeostasis. Our proposed future studies employing HIF2 knock out mice, model cell systems for HIF1 and HIF2, and the role of GSH in platelet function will be used to further explore the molecular mechanisms that regulate these complex pathways.

Substrate Loop in Glutathione Synthetase: Mutation Analysis

Glutathione (GSH), a small tripeptide, is a required cellular antioxidant. Cellular GSH is synthesized by two ATP‐dependent enzymes. The first enzyme, gamma‐glutamylcysteine synthetase, ligates glutamate and cysteine to form gamma‐glutamylcysteine. The second enzyme, glutathione synthetase (GS), ligates glycine to gamma‐glutamylcysteine to form GSH. Human GS, a homodimer, contains a conserved eleven amino acid residue loop region (266‐FRDGYMPRQYS‐276) called the Substrate Loop (S‐loop). In order to study the role of the conserved amino acid residues, several GS mutant enzymes, such as Gly269Val and Tyr270Phe, within this region were prepared using PCR site‐directed mutagenesis. These were expressed, purified and assayed for activity. Our findings show that mutations at Gly269 cause a significant decrease in GS activity (< 5% wild type). (Supported by Welch Foundation Chemistry Department grant; TWU A&S Research Development Fund (MEA).)

C‐Terminal Residues of Glutathione Synthetase

Glutathione (GSH:γ‐L‐Glutamyl‐L‐cysteinylglycine) is an important antioxidant found in living cells. Low cellular levels of GSH are associated with diseases such as stroke, AIDS and diabetes. Glutathione Synthetase (GS) catalyses the final ATP‐dependent step to form GSH. Human GS is the only mammalian member of the ATP‐grasp super‐family (an important class of amine carboxylate ligases) whose function is known; other family members are involved in nucleotide and cell wall synthesis. Each monomer of the dimeric GS has an active site, and GS displays negative cooperativity. There are three highly conserved loop regions (G‐,A‐,S‐loops). Higher eukaryotes have high A‐loop (alanine‐rich) identity (∼90%), while there is less identity in lower eukaryotes and prokaryotes (∼45%). The thirteen residue (Ile454‐Ala466) A‐loop is near the C‐terminal portion of GS and the glycine binding site. Several residues of the A‐loop were computationally modeled, mutated by site‐directed mutagenesis, and the mutant GS enzymes purified and assayed to characterize their role in GS function. We studied several GS mutants and found Glu 445 Arg and His 456 Ala enzymes have lowered activity (∼30% and ∼50%, respectively) compared to wild type GS. This A‐loop is important for GS activity. (Supported by a Welch Foundation Grant (Chem. Dept. TWU), U.S. Dept. Ed (CASCaM UNT, T.R.C.), Faculty Research Grant UNT (TRC).