Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes.
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ABSTRACT:
SUBMITTER: Clonis YD
PROVIDER: S-EPMC1162204 | biostudies-other | 1980 Oct
REPOSITORIES: biostudies-other
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Project description: Not available
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Project description:Azide-containing compounds have broad utility in organic synthesis and chemical biology. Their use as powerful tools for the labeling of biological systems in vitro has enabled insights into complex cellular functions. To date, fluorogenic azide-containing compounds have primarily been employed in the context of click chemistry and as sensitive functionalities for hydrogen sulfide detection. Here, we report an alternative use of this functionality: as fluorogenic probes for the detection of depleted oxygen levels (hypoxia). Oxygen is imperative to all life forms, and probes that enable quantification of oxygen tension are of high utility in many areas of biology. Here we demonstrate the ability of an azide-based dye to image hypoxia in a range of human cancer cell lines. We have found that cytochrome P450 enzymes are able to reduce these probes in an oxygen-dependent manner, while hydrogen sulfide does not play an important role in their reduction. These data indicate that the azide group is a new bioreductive functionality that can be employed in prodrugs and dyes. We have uncovered a novel mechanism for the cellular reduction of azides, which has implications for the use of click chemistry in hypoxia.
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Project description:NAADP has been shown to be a potent calcium-releasing second messenger in a wide variety of cell types to date. However, research has been hampered by a lack of pharmacological agents, with which to investigate NAADP-induced calcium release, and by the molecular identity of its cellular target protein being unknown. In the present paper, the sea urchin egg model was used to investigate whether triazine dyes, which can act as nucleotide mimetics, can bind to the NAADP receptor, induce Ca(2+) release and be used for affinity chromatography of the receptor. Indeed, all the triazine dyes tested (Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Green 5 (RG5), Cibacron Blue 3GA and Reactive Yellow 86) displayed micromolar affinities, except for Reactive Orange 14. Furthermore, unlike NAADP, RR120, RG19 and RG5 did not bind in an irreversible manner. The compound that displayed the highest affinity, RR120, was tested in a (45)Ca(2+) efflux assay. This compound released Ca(2+) via the NAADP receptor, as shown by the ability of subthreshold NAADP concentrations to inhibit this release. Furthermore, heparin and ruthenium red were unable to block RR120-induced Ca(2+) release. We have also shown that RG5 and RG19, immobilised on resins, retain the ability to bind to the receptor, and that this interaction can be disrupted by high salt concentrations. As a proof of principle, we have shown that this can be used to partially purify the NAADP receptor by at least 75-fold. In conclusion, triazine dyes interact with the NAADP receptor, and this could be exploited in future to create a new generation of pharmacological tools to investigate this messenger and, in combination with other techniques, to purify the receptor.
| S-EPMC1575193 | biostudies-other
Project description:1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.
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Project description:The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.
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Project description:Cu-catalyzed 1,3-dipolar cycloaddition of ethyl 2-azidoacetate to iodobuta-1,3-diynes and subsequent Sonogashira cross-coupling were used to synthesize a large series of new triazole-based push-pull chromophores: 4,5-bis(arylethynyl)-1H-1,2,3-triazoles. The study of their optical properties revealed that all molecules have fluorescence properties, the Stokes shift values of which exceed 150 nm. The fluorescent properties of triazoles are easily adjustable depending on the nature of the substituents attached to aryl rings of the arylethynyl moieties at the C4 and C5 atoms of the triazole core. The possibility of 4,5-bis(arylethynyl)-1,2,3-triazoles' application for labeling was demonstrated using proteins and the HEK293 cell line. The results of an MTT test on two distinct cell lines, HEK293 and HeLa, revealed the low cytotoxicity of 4,5-bis(arylethynyl)triazoles, which makes them promising fluorescent tags for labeling and tracking biomolecules.
| S-EPMC9147796 | biostudies-literature
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Project description:A series of chemically-defined adenosine phosphate ligands attached to Sepharose 4B were used as active-site probes in studying the interaction of enzymes with their coenzymes and substrates and to test the suitability of these matrices for ;general ligand' affinity chromatography. Nicotinamide nucleotide-dependent dehydrogenases were used as models to test this methodology. Elution from these columns by NAD(+) and/or AMP gradients (in the presence or the absence of substrates and/or nicotinamide mononucleotide) was consistent with: (1) the compulsory ordered addition of substrates to lactate and malate dehydrogenase; (2) the necessity for the NMN moiety of NAD(+) to bind to these enzymes before the substrate; and illustrated: (3) that the binding of these two hydrogenases to these columns compared very well with the published three-dimensional models for these enzymes and (4) that separation of mixtures of dehydrogenases depended on the choice of matrix and displacing ion and whether any additions (e.g. substrates) were made to the gradients used. These techniques were used to purify UDP-glucose dehydrogenase from a crude starting material on a phosphate-linked UDP (or ADP) matrix. The binding of this enzyme to these two columns was not consistent with either an ordered or random addition of substrates and suggested a more complex mechanism.
| S-EPMC1168184 | biostudies-other
Project description:Activation-induced cytidine deaminase (AID) generates U:G mismatches in Ig genes that can be converted into untemplated mutations during somatic hypermutation or DNA double-strand breaks during class switch recombination (CSR). Null mutations in UNG and MSH2 demonstrate the complementary roles of the base excision repair (BER) and mismatch repair pathways, respectively, in CSR. Phosphorylation of AID at serine 38 was previously hypothesized to regulate BER during CSR, as the AID phosphorylation mutant, AID(S38A), cannot interact with APE1, a BER protein. Consistent with these findings, we observe a complete block in CSR in AIDS38A/S38AMSH2-/- mouse B cells that correlates with an impaired mutation frequency at 5'Sμ. Similarly, somatic hypermutation is almost negligible at the JH4 intron in AIDS38A/S38AMSH2-/- mouse B cells, and, consistent with this, NP-specific affinity maturation in AIDS38A/S38AMSH2-/- mice is not significantly elevated in response to NP-CGG immunization. Surprisingly, AIDS38A/S38AUNG-/- mouse B cells also cannot complete CSR or affinity maturation despite accumulating significant mutations in 5'Sμ as well as the JH4 intron. These data identify a novel role for phosphorylation of AID at serine 38 in mismatch repair-dependent CSR and affinity maturation.
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Project description:Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER, but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA, but also neighboring linker DNA and nucleosome-free regions (NFRs). Furthermore, our data indicate that RSC plays a direct role in transcription coupled-NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.
2021-04-12 | GSE161930 | GEO