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

Volohonsky, Gloria; Tuby, Chen N.Y.H.; Porat, Noga; Wellman-Rousseau, Maria; Visvikis, Athanase; Leroy, Pierre; Rashi, Sharon; Steinberg, Pablo; Stark, Avishay‐Abraham

doi:10.1016/s0009-2797(02)00017-0

Cited by 35 publications

(24 citation statements)

References 27 publications

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“…c-ECS (EC 6.3.2.2) activity c-Glutamylcysteine synthetase activity assay was adapted from the indirect method of Volohonsky et al (2002), which utilizes the coupled reaction of pyruvate kinase (PK) and lactate dehydrogenase (LDH) to determine the rate of formation of ADP by c-ECS through the oxidation of NADH. The reaction mixture contained 0.1 M Tris-HCl buffer, pH 8, 150 mM KCl, 2 mM EDTA, 20 mM MgCl 2 , 5 mM ATP, 2 mM phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L-a-aminobutyrate, 0.2 mM NADH, 7 U ml -1 PK and 10 U ml -1 LDH.…”

Section: Measurements Of Gsh and Gssgmentioning

confidence: 99%

Influence of GA3, sucrose and solid medium/bioreactor culture on in vitro flowering of Spathiphyllum and association of glutathione metabolism

Dewir

Chakrabarty

Ali

et al. 2007

Plant Cell Tiss Organ Cult

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Hormonal control of flower induction and inflorescence development in vitro was investigated in Spathiphyllum. The effects of gibberellic acid (GA 3 ) and sucrose on inflorescence development were studied in plantlets regenerated in tissue culture. GA 3 was mandatory for the shift from the vegetative to the reproductive stage. The effect of sucrose concentration on inflorescence bud development was studied in plantlets cultured in MS medium supplemented with 10 mg l -1 GA 3 . Sucrose concentration at 3 or 6% induced inflorescence development in, respectively, 83-85% of the plantlets. The effect of GA 3 and sucrose on inflorescence differentiation and development were also recorded in liquid culture using air-lift bioreactor. The best response was found in the same medium which was standardized as an optimum for solid culture, but the results were better than solid culture. In order to study the relationship between glutathione (GSH) and flowering, we also measured the oxidized and reduced GSH content in leaves throughout the culture period on 2 weeks interval. The GSH accumulation was more after 4 weeks until 6 weeks in GA 3 treated plantlets. Similarly, glutathione reductase which is involved in the recycling of reduced GSH providing a constant intracellular level of GSH, was also higher in GA 3 treated plantlets. The transient increase in GSH contents also correlated with the changes in measured c-glutamylcysteine synthetase (c-ECS) activity over the same period. The antioxidant enzyme activity in GA 3 treated plantlets also suggests that the plants suffered increased oxidative stress during the period of GA 3 treatment which subsequently increases GSH synthesis through activation of c-ECS and this promotes flowering by increasing endogenous GSH.

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Section: Measurements Of Gsh and Gssgmentioning

confidence: 99%

Influence of GA3, sucrose and solid medium/bioreactor culture on in vitro flowering of Spathiphyllum and association of glutathione metabolism

Dewir

Chakrabarty

Ali

et al. 2007

Plant Cell Tiss Organ Cult

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show abstract

“…c-GCS activity (c-glutamylcysteine synthetase) c-glutamylcysteine synthetase activity assay was adapted from the indirect method of Volohonsky et al (2002), which utilizes the coupled reaction of pyruvate kinase (PK) and lactate dehydrogenase (LDH) to determine the rate of formation of ADP by c-GCS through the oxidation of NADH. Reaction mixture contained 0.1 M Tris-HCl buffer, pH 8, 150 mM KCl, 2 mM EDTA, 20 mM MgCl 2 , 5 mM ATP, 2 mM phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L-a-aminobutyrate, 0.2 mM NADH, 7 U ml À1 PK, and 10 U ml À1 LDH.…”

Section: Determination Of Antioxidant Enzymes Activitymentioning

confidence: 99%

Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor

Shohael

Ali²,

Hahn³

et al. 2007

Plant Cell Tiss Organ Cult

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Compared to non-embryogenic callus, proembryonic mass, globular, and heart-shaped embryos of Eleutherococcus senticosus had higher levels of endogenous reduced glutathione (GSH). GSH content declined during the course of the embryo development (torpedo and cotyledon). Similarly, glutathione reductase that is involved in the recycling of GSH providing a constant intracellular level of GSH was also higher in globular and heartshaped embryos. The transient increase in GSH contents also correlated with the changes in measured c-glutamylcysteine synthetase activity over the same period. The endogenous levels of oxidized glutathione showed similar trend during development of the somatic embryos, whereas it declined in maturing somatic embryos. A pronounced increase in glutathione-S-transferase, glutathione peroxidase, catalase, and guaiacol peroxidase activity was observed during somatic embryo maturation. Ascorbate-glutathione cycle enzymes (ascorbate peroxidase; dehydroascorbate reductase and monodehydroascorbate reductase) activities also induced indicated that antioxidant enzymes played an important role during embryo development. These results suggested that the coordinated up-regulations of the antioxidant enzymes and glutathione redox system provide protection during somatic embryo development in E. senticosus. Antioxidant responses through alterations of the glutathione redox systems, have been described in the present studies have a significant role in somatic embryo development.

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“…Recent technological advances in HPLC, and appropriately designed preincubation strategies, have enabled us to directly (i.e., by direct determination of γ-GC) investigate the effect of dephosphorylation/phosphorylation cycles on the activity of GCL [31,39]. The results shown in Fig.…”

Section: Preincubation Of Protein Preparations Under Phosphorylating mentioning

confidence: 99%

Mechanisms of γ-glutamylcysteine ligase regulation

Toroser

Yarian

Orr

et al. 2006

Biochimica et Biophysica Acta (BBA) - General Subjects

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The principal objective of this study was to investigate the mechanisms regulating the activity of γ-glutamylcysteine ligase (GCL; EC 6.3.2.2), the rate limiting enzyme in glutathione biosynthesis. Two phylogenetically divergent species, mouse and the fruitfly, Drosophila melanogaster were used to test the hypothesis that reversible protein phosphorylation and pyridine dinucleotide phosphate dependent allostery regulate GCL activity. GCL was almost completely inhibited under phosphorylating conditions, involving preincubations with MgATP and endogenous protein kinases. Maximal GCL inhibitions of 94%, 77%, 85%, 87%, 83%, 95% and 89% occurred, respectively, in mouse cerebellum, hippocampus, brainstem, striatum, cortex and heart, and Drosophila. These changes in GCL activity were detected using saturating levels of substrates, suggesting that V max was dramatically affected, whereas K m values showed no differences. In vitro activation of GCL, presumably due to dephosphorylation, was blocked by inhibitors of protein phosphatases, suggesting that GCL exists in vivo as a mixture of phosphorylated and dephosphorylated forms. The reversibility of the dephosphorylation-dependent activation was indicated by the time-dependent inactivation of the in vitro activated Drosophila GCL, by preincubation with MgATP. NADPH increased maximal GCL activity by up to 93%, whereas several other nucleotide analogues did not, thereby demonstrating specificity. Kinetic analysis using Hanes-Woolf replots of initial velocity data suggested that the NADPH-dependent stimulation of GCL activity is brought about by a change in the maximal activity, V max , rather than changes in substrate affinity. Results of this study suggest that mechanisms of modulation of eukaryotic GCL enzymes may include specific binding of ligands such as pyridine dinucleotide phosphates and reversible protein phosphorylation.

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

Cited by 35 publications

References 27 publications

Influence of GA3, sucrose and solid medium/bioreactor culture on in vitro flowering of Spathiphyllum and association of glutathione metabolism

Influence of GA3, sucrose and solid medium/bioreactor culture on in vitro flowering of Spathiphyllum and association of glutathione metabolism

Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor

Mechanisms of γ-glutamylcysteine ligase regulation

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