The glutathione-deficient, cadmium-sensitive mutant, cad2–1 , of Arabidopsis thaliana is deficient in γ-glutamylcysteine synthetase

This paper reports that the glutathione (GSH)-deficient mutant, cad2–1 , of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, γ-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, γ-glutamylcysteine. In vitro enzyme assays showed that the cad2–1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA , identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2–1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2–1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2–1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.     

γ-Glutamylcysteine synthetase and γ-glutamyl transferase as differential enzymatic sources of γ-glutamylpeptides in mice

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography–mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.     

Responses to cadmium in leaves of transformed poplars overexpressing Γ-glutamylcysteine synthetase

Poplars overexpressing a bacterial Γ -glutamylcysteine synthetase ( Γ -ECS) in the cytosol (lines ggs11 and ggs28) had a 30-fold increase in foliar Γ -ECS activity relative to untransformed controls. Foliar Γ -glutamylcysteine ( Γ -EC) was increased by 10-fold while foliar glutathione accumulation increased by up to 3.5-fold in the leaves of the transformants. Untransformed and transformed poplars were grown with different soil concentrations of cadmium (0–1100 μg g⁻¹ soil) for 2 weeks. Cadmium accumulated in the leaves of both transformed and untransformed poplars and growth was inhibited. Growth inhibition and foliar cadmium accumulation were greatest at the highest soil cadmium concentrations in all lines. Exposure to cadmium enhanced the foliar cysteine, Γ -EC and glutathione pools in all lines but less glutathione was present in the leaves of the untransformed controls than the transformants under all growth conditions. Cadmium-induced changes in the activities of malic enzyme, isocitrate dehydrogenase and guaiacol peroxidase were less pronounced in the leaves of the transformed poplars overexpressing Γ -ECS than in the untransformed controls. Glutamate dehydrogenase and glutathione reductase activities were unchanged by exposure to cadmium. We conclude that overexpression of Γ -ECS activity and foliar glutathione accumulation in transformed poplar allows greater tissue cadmium accumulation but has only a marginal effect on cadmium tolerance in poplar.     

Conversion of γ-Hydroxybutyrate to γ-Aminobutyrate In Vitro

[3H]gamma-Hydroxybutyric acid [( 3H]GHB) at physiological concentration incubated with brain slices in Krebs-Ringer medium produced [3H]gamma-aminobutyric acid [( 3H]GABA). This compound was identified by its Rf values on thin-layer chromatograms and by analysis of the dansyl derivatives of the free amino acid fraction. No labelled glutamate could be detected. Brain slices incubated with labelled glutamate and nonradioactive GHB generated labelled 2-oxoglutarate, suggesting that gamma-aminobutyrate-2-oxoglutarate transaminase (GABA-T) is involved in catalyzing this reaction. Furthermore, specific inhibitors of GABA-T blocked the production of labelled GABA from labelled GHB and of labelled 2-oxoglutarate from labelled glutamate. Transformation of [3H]GHB into [3H]GABA was not inhibited by malonate, demonstrating that the succinate-linked pathway is not involved in the generation of GABA. The kinetic characteristics of the multienzyme system involved in GHB degradation studied in vitro are compatible with the production of GABA in vivo.     

Accumulation of γ‐aminobutyric acid in nodulated soybean in response to drought stress

Nitrogen fixation and nodule permeability to O₂ diffusion are decreased by drought stress. Since γ‐aminobutyric acid (GABA) synthesis is rapidly stimulated by a variety of stress conditions including hypoxia, it was hypothesized that decreased O₂ availability in nodules stimulates glutamate decarboxylase (GAD) activity (EC 4.1.1.15), thereby resulting in GABA accumulation. First, the amino acid composition of xylem sap was determined in plants subjected to soil water deficits. While the xylem sap concentration of several amino acids increased when the plant was subjected to a water deficit, the greatest increase was in GABA. GABA accumulation was examined in response to stress induced by hypoxia or the addition of polyethylene glycol (PEG) to the nutrient solution. The exposure of soybean nodules to hypoxia for 6 h enhanced the GABA concentration by 6‐fold, but there was no change in GABA concentration in response to the PEG treatment. No major changes in the in vitro GAD activity were measured in nodule cytosol or bacteroids. The present data do not support the hypothesis that decreased nodule O₂ permeability and a resulting O₂ deprivation inside nodules may stimulate in vitro GAD activity and thus GABA accumulation. However, the data could indicate a possible effect of hypoxia and drought stress on the in vivo activity of GAD.     

Influence of cadmium on the production of γ-glutamylcysteine peptides and enzymes of nitrogen assimilation in Zea mays seedlings

We examined the effect of cadmium (Cd) additions on a GDH1-null line of maize and its wild-type isogenic sibling. Addition of Cd increases the synthesis of metallothioneines which are glutamate- and cysteine-rich peptides. We predicted a reduced synthesis of γ-glutamylcysteine (γEC) peptides in the mutant relative to the wild type if glutamate dehydrogenase (GDH) was limiting the drainage of carbon from the tricar-boxylic acid cycle (TCAC). In our experiments there were similar increases in levels of γEC peptides in both mutant and wild-type seedlings in response to Cd. There was a marked increase in the phosphoenolpyruvate carboxylase (PEPcase) polypeptide and in one of the polypeptide bands of glutamine synthetase in both mutant and wild-type seedlings. However, no change was seen in the polypeptide levels of GDH or glutamate synthase (GOGAT). Thus, in contrast to PEPcase, an enhanced carbon drain from the TCAC in response to Cd exposure does not require enhanced levels of either GDH or GOGAT polypeptides.     

Genetic and chemical analyses reveal that trypanothione synthetase but not glutathionylspermidine synthetase is essential for Leishmania infantum

Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps^−/− knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys^−/−/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.     

Distribution of γ-Tubulin in Higher Plant Cells: Cytosolic γ-Tubulin is Part of High Molecular Weight Complexes

Abstract: γ-Tubulin is a protein found in all eukaryotic cells, where it plays a key role in the nucleation of microtubules. In higher plant cells, γ-tubulin is localized at the nuclear surface, a known microtubule-organizing centre, and is codistributed with all microtubule arrays. Functions of plant γ-tubulin remain to be determined. This study describes some properties of higher plant γ-tubulin. The overall level of γ-tubulin was constant during the cell cycle in synchronized tobacco BY-2 cells. Biochemical analysis of the subcellular distribution of γ-tubulin in maize cells revealed that, in contrast with animal γ-tubulin, plant γ-tubulin is mainly associated with endomembranes. We showed for the first time that the pool of soluble cytosolic γ-tubulin contained two main γ-tubulin complexes. γ-tubulin, Hsp70 and TCP1-related proteins might interact in a small complex of 750 kDa. A second γ-tubulin complex, larger than 1500 kDa was purified. The protein profile of this large complex was very similar to animal γ-tubulin complexes. The putative functions of these two complexes in plant microtubule nucleation are discussed.     

The Extraneural Distribution of γ-Hydroxybutyrate

Abstract— γ-Hydroxybutyrate has been found to be widely distributed in both neural and extraneural tissues in the rat. The kidney and brown fat have more than 10 times higher concentrations of y-hydroxybutyrate than does the brain. This observation suggests that γ-hydroxybutyrate may participate in the metabolism of many organs, and that GABA may not be the precursor in extraneural tissues.     

The appearance of α, β and γ crystallins in an anophthalmic strain of mice

Abstract. A certain percentage of congenitally anophthalmic mouse embryos have the ability to generate small lens vesicles that have previously been shown to produce alpha crystallin at 13-day gestation. Further immunohistological analysis of 13- and 15-day-gestation anophthalmia embryos indicates that beta crystallin is present in those 13-day embryos which have lens vesicles with lens-fiber formation. Also, 15-day embryos with lenses demonstrating fiber elongation can produce both beta and gamma crystallins. The conclusion is drawn that the genetic potential to produce at least three characteristic biochemical markers of normal lens differentiation is present in the anophthalmia mutant. The spatial distribution patterns of the crystallins in normal and anophthalmia embryos were similar. However, there appeared to be a transposition in the temporal appearance of beta and gamma crystallins in the anophthalmia mutant. Optic cups and associated lenses in 15-day anophthalmia specimens were much smaller than those in controls. The optic and lens rudiments in these anophthalmia embryos were fairly proportional in size, which indicates that some degree of allometric growth compensation had occurred during the course of development. This ability for differential growth compensation in the mouse eye appears to be restricted to the predifferentiative stages of eye formation.     

Functional analysis of γ-carotene ketolase involved in the carotenoid biosynthesis of Deinococcus radiodurans

Deinococcus radiodurans strain R1 synthesizes a unique ketocarotenoid product named deinoxanthin. The detailed steps involved in the biosynthesis of deinoxanthin remain unresolved. A carotene ketolase homologue encoded by dr0093 was inactivated by gene mutation to verify its function in the native host D. radiodurans . Analysis of the carotenoids in the resultant mutant R1ΔcrtO demonstrated that dr0093 encodes γ-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position of γ-carotene derivatives to form ketolated carotenoids. The mutant R1ΔcrtO became more sensitive to H₂O₂ treatment than the wild-type strain R1, indicating that the C-4 keto group is important for the antioxidant activity of carotenoids in D. radiodurans . Carotenoid extracts from mutant R1ΔcrtO exhibited lower 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the wild-type strain R1. The enhanced antioxidant ability of ketocarotenoids in D. radiodurans might be attributed to its extended conjugated double bonds and relative stability by the C-4 keto group substitution.     

Effects of Anticonvulsants on the Formation of γ-Hydroxybutyrate from γ-Aminobutyrate in Rat Brain

Abstract: The conversion of γ-aminobutyrate (GABA) via succinic semialdehyde to γ-hydroxybutyrate has been examined in rat brain homogenates. A number of anticonvulsants, including sodium valproate and phenobarbitone, inhibited this metabolic pathway. These results are interpreted in the light of the characteristics of aldehyde reductases known to reduce succinic semialdehyde.     

The tyrosine-6 hydroxyl of γδ resolvase is not required for the DNA cleavage and rejoining reactions

Site-specific recombinases of the resolvase and DNA invertase family all contain a tyrosine residue close to the N-terminus, and four residues away from a serine that has been implicated in catalysis of DNA strand breakage and reunion. To examine the role of this tyrosine in recombination, we have constructed a mutant of γδ resolvase in which the tyrosine (residue 6) is replaced by phenytalanine. Characterization of the Y6F mutant protein in vitro indicated that although it was highly defective in recombination, it could cleave ONA at the cross-over site, form a covalent resolvase-DNA complex and rejoin the cleaved cross-over site (usually restoring the parental site). These data rule out a direct role of the Tyr-6 hydroxyl as the nucleophile In the DNA cleavage reaction and strengthen the conclusion that this nucleophile is the nearby invariant serine residue, Ser-10. We conclude that Tyr-6 is essential for fully coordinated strand cleavage and exchange, but is dispensable for individual strand cleavage and religation reactions.     

Identification and characterization of a strain-dependent cystathionine β/γ-lyase in Lactobacillus casei potentially involved in cysteine biosynthesis

The trans -sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans -sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans -sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei , which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli . The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds.     

Effect of L-3,4-dihydroxyphenylalanine, ornithine and γγ-aminobutyric acid on winter wheat resistance to grain aphid

The association between the concentrations of L-3,4-dihydroxyphenylalanine (L-DOPA), ornithine, and γ-aminobutyric acid (GABA) in winter wheat cultivars with their antibiotic resistance to the grain aphid Sitobion avenae (F.) (Hom., Aphididae) was studied. The antibiosis was measured under field conditions using the intrinsic rate of natural increase ( r _m ). The content of the studied non-protein amino acids was estimated in flag leaf at the beginning of anthesis growth stage. Highly significant negative correlations were found between the r _m values and concentrations of L-DOPA (r = −0.937) and ornithine (r = −0.907), but not with GABA (r = −0.363). The contribution of these allelochemicals to the resistance of the plants to insects is discussed.     

Bacterial γ-glutamyltranspeptidases: Comparison of Escherichia coli and Pseudomonasγ-glutamyltranspeptidase

Serpulina (Treponema) hyodysenteriae strain A-1 partially purified rRNA, labelled with photobiotin, was used as a non-radioactive probe to identify the rRNA gene restriction patterns of S. hyodysenteriae strains and other spirochetes. Sau3A restriction enzyme digests resulted in similar rRNA gene restriction patterns in S. hyodysenteriae strains from five different countries. Some S. hyodysenteriae strains could be differentiated by variations in their rRNA gene restriction patterns after cleavage of DNA by restriction enzymes SspI or BglII. S. innocens and Treponema succinifaciens, non-pathogenic pig intestinal spirochetes, had rRNA gene restriction patterns that differed markedly from the S. hyodysenteriae patterns, and from each other.     

The wheat TaGBF1 gene is involved in the blue‐light response and salt tolerance

Light and abiotic stress both strongly modulate plant growth and development. However, the effect of light‐responsive factors on growth and abiotic stress responses in wheat (Triticum aestivum) is unknown. G–box binding factors (GBFs) are blue light‐specific components, but their function in abiotic stress responses has not been studied. Here we identified a wheat GBF1 gene that mediated both the blue light‐ and abiotic stress‐responsive signaling pathways. TaGBF1 was inducible by blue light, salt and exposure to abscisic acid (ABA). TaGBF1 interacted with a G–box light‐responsive element in vitro and promoted a blue‐light response in wheat and Aradidopsis thaliana. Both TaGBF1 over‐expression in wheat and its heterologous expression in A. thaliana heighten sensitivity to salinity and ABA, but its knockdown in wheat conferred resistance to high salinity and ABA. The expression of AtABI5, a key component of the ABA signaling pathway in A. thaliana, and its homolog Wabi5 in wheat was increased by transgenic expression of TaGBF1. The hypersensitivity to salt and ABA caused by TaGBF1 was not observed in the abi5 mutant background, showing that ABI5 is the mediator in TaGBF1‐induced abiotic stress responses. However, the hypersensitivity to salt conferred by TaGBF1 is not dependent on light. This suggests that TaGBF1 is a common component of blue light‐ and abiotic stress‐responsive signaling pathways.