A versatile bacterial enzyme: l-methionine γ-lyase

The properties and application of l-methionine γ-lyase [methioninase, l-methionine methanethiol-lyase (deaminating), EC 4.4.1.11], a pyridoxal 5′-phosphate enzyme, purified from Pseudomonas putida and Aeromonas sp. are presented. The enzyme has multicatalytic functions: it catalyses α,γ-elimination and γ-replacement reactions of l-methionine and its analogues (e.g. ethionine, homocysteine, O-acetylhomoserine and selenomethionine), α,β-elimination and β-replacement reactions of l-cysteine and its analogues (e.g. S-methylcysteine, O-acetylserine and Se-methylselenocysteine), deamination and γ-addition of vinylglycine, and deuterium labelling at the α and β positions of l-methionine and other straight-chain l-amino acids. These reactions are applicable to the synthesis of various optically active sulphur and selenium amino acids, preparation of deuterium or tritium labelled l-amino acids, and determination of sulphur amino acids. In addition, the enzyme shows potent anti-neoplastic activity.     

A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase

γ-Glutamylcyclotransferase (GGCT) is a component of the γ-glutamyl cycle and specifically cleaves γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline. The action of GGCT in glutathione synthetase-deficient patients results in the accumulation of high levels of 5-oxoproline. In addition, GGCT may be a biomarker in some cancers. GGCT inhibitors, therefore, may be of value in the treatment of glutathione synthetase deficiency or in the elucidation of the role of GGCT in cancer cells. Published GGCT assays are not suitable for high-throughput screening for inhibitory molecules. We have developed a fluorescence-based 96-well plate assay for the determination of the rate of γ-glutamylcysteine cleavage by GGCT. After the reaction, the residual γ-glutamylcysteine is determined fluorometrically after derivatization with 2,3-naphthalenedicarboxaldehyde. The method has sufficient sensitivity to detect low-affinity competitive inhibitors and, as a result of its simplicity and microtiter plate format, can be readily used in high-throughput inhibitor screens.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.     

A spelt-specific γ-gliadin gene: discovery and detection

Polymerase chain reaction (PCR) primers GAG5 and GAG6 were designed based on published γ-gliadin gene sequences and applied to 35 cultivars of closely related spelt (Triticum spelta L.) and hexaploid wheat (T. aestivum L.). Eight tetraploid durum wheat (T. durum Desf.) cultivars were included in the analysis. The obtained PCR products originated from two γ-gliadin genes which were mapped to homeologous chromosomes 1B and 1D and termed GAG56B and GAG56D, respectively. Two alleles of GAG56D differing in a 9-bp deletion/duplication and single nucleotide polymorphism were found. The 18 spelts tested and wheat cultivar ’Chinese Spring’ were discovered to carry a previously unknown γ-gliadin gene, while 16 wheat cultivars possessed its longer, already published allele. Two PCR-based detection systems for the diagnostic alleles were developed and applied. The occurrence of two alleles of GAG56B among the investigated durum wheats correlated with their expression of gluten quality markers γ-gliadins 42 or 45. Received: 10 March 1999 / Accepted: 17 March 1999     

Increased γ-H2A.X Intensity in Response to Chronic Medium-Dose-Rate γ-Ray Irradiation

Background

The molecular mechanisms of DNA repair following chronic medium-dose-rate (MDR) γ-ray-induced damage remain largely unknown.

Methodology/Principal Findings

We used a cell function imager to quantitatively measure the fluorescence intensity of γ-H2A.X foci in MDR (0.015 Gy/h and 0.06 Gy/h) or high-dose-rate (HDR) (54 Gy/h) γ-ray irradiated embryonic fibroblasts derived from DNA-dependent protein kinase mutated mice (scid/scid mouse embryonic fibroblasts (scid/scid MEFs)). The obtained results are as follows: (1) Automatic measurement of the intensity of radiation-induced γ-H2A.X foci by the cell function imager provides more accurate results compared to manual counting of γ-H2A.X foci. (2) In high-dose-rate (HDR) irradiation, γ-H2A.X foci with high fluorescence intensity were observed at 1 h after irradiation in both scid/scid and wild-type MEFs. These foci were gradually reduced through de-phosphorylation at 24 h or 72 h after irradiation. Furthermore, the fluorescence intensity at 24 h increased to a significantly greater extent in scid/scid MEFs than in wild-type MEFs in the G₁ phase, although no significant difference was observed in G₂/M-phase MEFs, suggesting that DNA-PKcs might be associated with non-homologous-end-joining-dependent DNA repair in the G₁ phase following HDR γ-ray irradiation. (3) The intensity of γ-H2A.X foci for continuous MDR (0.06 Gy/h and 0.015 Gy/h) irradiation increased significantly and in a dose-dependent fashion. Furthermore, unlike HDR-irradiated scid/scid MEFs, the intensity of γ-H2A.X foci in MDR-irradiated scid/scid MEFs showed no significant increase in the G₁ phase at 24 h, indicating that DNA repair systems using proteins other than DNA-PKcs might induce cell functioning that are subjected to MDR γ-ray irradiation.

Conclusions

Our results indicate that the mechanism of phosphorylation or de-phosphorylation of γ-H2A.X foci induced by chronic MDR γ-ray irradiation might be different from those induced by HDR γ-ray irradiation.     

Pore-forming scissors? A first structural glimpse of γ-secretase

Amyloid plaques, which are composed of amyloid-beta peptide (Abeta), signify Alzheimer's disease pathology. Secretases generate Abeta by processing the beta-amyloid precursor protein. gamma-Secretase, a complex comprising four different proteins, liberates Abeta from its precursor by intramembrane proteolysis. The first impression of the shape of gamma-secretase has recently been revealed by electron microscopy. It indicates a spherical transmembrane particle with an interior chamber that, presumably, accommodates its catalytic residues, and two openings that might be exit sites for the cleavage products.     

Resistance of Clostridium perfringens Type A Spores to γ-Radiation

The radiation resistance of the spores of a classical strain and of an atypical, heat-resistant strain of Clostridium perfringens was determined. Spores were produced in Ellner's and in a Trypticase broth medium. Approximately 10⁶ viable spores per milliliter were suspended in 0.06 m phosphate buffer and irradiated with γ rays from cobalt-60; the survivors were counted in Tryptone-yeast extract-agar by the Prickett-tube technique. Radiation D values for spores of the atypical strain in phosphate buffer and in cooked-meat broth were 0.23 and 0.30 Mrad, respectively, and the D value of the classical strain was 0.25 Mrad in phosphate buffer. Spores of the classical and atypical strains of C. perfringens type A are characterized by differences in heat resistance; yet, all strains tested demonstrated similar radiation resistance. Also, the spores were more resistant to ionizing radiation in cooked-meat broth than in phosphate buffer.     

Heterochromatin Controls γH2A Localization in Neurospora crassa

In response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called γH2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of γH2A followed by next-generation sequencing (ChIP-seq). γH2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal γH2A localization. In contrast, γH2A is not required for H3K9 methylation or DNA methylation. Normal γH2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. γH2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.     

A Unique γ-Glutamyltransferase from Fruiting Bodies of Tricholoma shimeji

The extract of terrestrial alga Nostoc commune Vauch. has high antioxidative activity. Our study on N. commune Vauch. resulted in the isolation of two β-ionone derivatives, nostocionone and 3-oxo-β-ionone, together with four indole alkaloids, scytonemin, reduced scytonemin, N-(p-coumaroyl)tryptamine, and N-acetyltryptamine. The structures of the isolated compounds were determined on the basis of 1D and 2D NMR and MS analyses. Among these isolates, nostocionone and reduced scytonemin demonstrated strong antioxidative activities which were assessed by using a β-carotene oxidation assay.     

A Taq1 γ-globin DNA polymorphism: an African-specific marker

Summary The allele frequency of a Taq 1 -globin gene restriction fragment length polymorphism (RFLP) is reported in ten population groups. In four African populations the 3.0 kb RFLP is common (50/132 {ie90-1} chromosomes), whereas it is completely absent in six European/Asian populations (0/277 {ie90-2} chromosomes). This Taq 1 RFLP is thus a specific African population marker.     

A sequential response to growth substances in coleoptiles from γ-irradiated wheat

Summary -irradiated wheat seed (500 kr) produces coleoptiles that grow without cell division or DNA synthesis. Apart from an initial 24-hr delay in growth, intact coleoptiles have a pattern of cell elongation similar to normal coleoptiles. The elongation of coleoptiles excised at a size of 2 mm, when the cells are small and just prior to entering a rapid elongation phase, is promoted by kinetin and gibberellic acid (GA₃). Elongation of coleoptiles excised at 8 mm, when the cells are larger and in the rapid elongation phase, is promoted by indoleacetic acid (IAA). This sequential response to growth substances in coleoptiles is remarkably similar to that in normal coleoptiles. The GA₃ response in excised coleoptiles is not inhibited by FUDR, confirming that DNA synthesis is not required for GA₃-induced elongation in coleoptiles.     

A Direct Interaction with NEDD1 Regulates γ-Tubulin Recruitment to the Centrosome

The centrosome is the primary microtubule organizing centre of the cell. γ-tubulin is a core component of the centrosome and is required for microtubule nucleation and centrosome function. The recruitment of γ-tubulin to centrosomes is mediated by its interaction with NEDD1, a WD40-repeat containing protein. Here we demonstrate that NEDD1 is likely to be oligomeric in vivo and binds directly to γ-tubulin through a small region of just 62 residues at the carboxyl-terminus of the protein. This carboxyl-terminal domain that binds γ-tubulin has a helical structure and is a stable tetramer in solution. Mutation of residues in NEDD1 that disrupt binding to γ-tubulin result in a mis-localization of γ-tubulin away from the centrosome. Hence, this study defines the binding site on NEDD1 that is required for its interaction with γ-tubulin, and shows that this interaction is required for the correct localization of γ-tubulin.     

Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase

The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.     

A ring for all: γ-tubulin-containing nucleation complexes in acentrosomal plant microtubule arrays

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Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis

The oligomeric states of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases (BlGGT and EcGGT) in solution have been investigated by analytical ultracentrifugation. The results showed that BlGGT has a sedimentation coefficient of 5.12S, which can be transformed into an experimental molecular mass of approximately 62,680Da. The monomeric conformation is conserved in EcGGT. SDS-PAGE analysis and cross-linking studies further proved that the autocatalytically processed BlGGT and EcGGT form a heterodimeric association. Unfolding analyses using circular dichroism and tryptophan emission fluorescence revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and EcGGT had a T(m) value of 59.5 and 49.2°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Chemical unfolding of BlGGT was independent to the pH value ranging from 5 to 10, whereas the pH environment was found to significantly influence the GdnHCl-induced denaturation of EcGGT. Both enzymes did not reactivate from the completely unfolded states, accessible at 6M GdnHCl. BlGGT was active in the presence of 4M NaCl, whereas the activity of EcGGT was significantly decreased at the high-salt condition. Taken together, these findings suggest that the biophysical properties of the homologous GGTs from two mesophilic sources are quite different.     

Chemical properties of guinea-pig immunoglobulins γ2G and γM

The isolation of guinea-pig immunoglobulins γ₁G, γ₂G and γM are described and methods for separating the polypeptide chains of each examined. The molecular weights, extinction coefficients and carbohydrate and amino acid compositions of the immunoglobulins and their constituent chains have been analysed. The findings provide a basis for further studies attempting to relate structural differences to distinct biological properties of guinea-pig immunoglobulins.