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PubMed1980

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium.

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Our of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium.

Azides↗

PubMed1981

In vitro synthesis of a mutagenic azide metabolite by cell-free bacterial extracts.

Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction not produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by L-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55 degrees C and had a pH optimum between 7-7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite.

Azides↗

PubMed2006

Comparison of IR and UV cavity ring-down spectroscopy detection of transient intermediates: pyrolysis of methyl azide to form methyleneimine.

Mid-infrared cavity ring-down spectroscopy (CRDS) has been employed in this study to examine the hydride stretching region of methyl azide and its pyrolysis product methyleneimine. The absorption spectrum of methyl azide over 2835-3085 cm(-1) was recorded, and the integrated absorption cross section was determined. The pyrolysis of methyl azide and subsequent production of methyleneimine was observed at various wavenumbers. Using IR CRDS, we were able to observe vibrational transitions of methyleneimine without interference from the methyl azide precursor. Our previous UV CRDS study showed that electronic transitions of methyleneimine overlapped with those of methyl azide. IR CRDS should thus be useful for the detection of polyatomic transient intermediates without interference from precursors.

Azides↗

PubMed2003

Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine.

In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1O2) we found that azide, usually used as 1O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661-665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed.

Antioxidants↗

PubMed1997

Inhibition of etoposide-induced apoptotic events by azide.

Azide is reported to be an inducer of necrotic rather than apoptotic cell death. Using various parameters of cell death, we demonstrate here that azide is capable of completely inhibiting apoptosis induced by VP-16/etoposide. Azide was found systematically to protect Jurkat cells against VP-16-induced effects as determined by an array of biochemical and morphological parameters. The cells were able partially to recover proliferative activity following removal of the drugs. We conclude that azide inhibits apoptosis induced by VP-16 at an early point in the apoptotic pathway and that inhibition of apoptosis might be the mechanism of necrosis-induction by azide.

Antineoplastic Agents, Phytogenic↗

PubMed1996

Peroxidatic degradation of azide by catalase and irreversible enzyme inactivation.

A study of the azide reaction with bovine liver catalase in presence of hydrogen peroxide has been performed, using conventional UV-visible spectrometry and activity measurements. Compound III and NO-ferrocatalase were the predominant forms of the enzyme observed in air and under nitrogen, respectively. A reaction scheme for peroxidatic degradation of azide by catalase is proposed. Accordingly, accumulation of Compound III is the main factor responsible for the reversible inhibition of 'catalatic' activity by azide, while formation of a complex between native catalase and azide has a negligible effect. Catalase is irreversibly inactivated by prolonged exposure to high levels of H2O2 and azide. The latter involves cleavage of the prosthetic group with liberation of the heme iron. Both in air and under nitrogen, generation of azidyl radicals seems to play a minor role in the irreversible inactivation process.

Animals↗

PubMed1994

Use of aryl azide cross-linkers to investigate protein-protein interactions: an optimization of important conditions as applied to Escherichia coli RNA polymerase and localization of a sigma 70-alpha cross-link to the C-terminal region of alpha.

In an effort to better understand protein-protein photoaffinity cross-linking using aryl azides, we have tested a number of factors influencing the cross-linking of the sigma 70 subunit of Escherichia coli RNA polymerase to core RNA polymerase. These factors include the effect of the incubations necessary for the derivatization of the protein on enzyme activity, the effect of overhead lighting on azide stability, the effect of reducing agents on azide stability, aggregation of the derivatized protein, and a comparison of two types of aryl azide cross-linkers, N-[(5-azido-2-nitrobenzoyl)oxy]succinimide (ANB-NOS) and (N-hydroxysuccinimidyl)-4-azidosalicylic acid (NHS-ASA). We found that derivatization proceeds effectively in a buffer similar to the buffer used during protein purification, that overderivatization can cause protein aggregation, that room lighting does not appreciably destroy aryl azides, and that 0.1 mM DTT is a better choice of reducing agent than 5 mM 2-mercaptoethanol. The cross-link products were separated by SDS gel electrophoresis and identified on Western blots by cross-reactivity with monoclonal antibodies to the individual subunits of RNA polymerase. In agreement with previous work (Coggins et al., 1977; Hillel & Wu, 1977), it was possible to cross-link sigma 70 to all three of the subunits of RNA polymerase. With a combination of gel analysis, chemical cleavage, and immunodetection, it is possible to demonstrate that sigma 70 cross-links to the alpha subunit between residues 209 and 329.

Azides↗

PubMed1976

Anomalous azide binding to metmanganomyoglobin.

The reaction of metmanganomyoglobin (MnIIIMb) with azide presents a novel pattern with direct evidence for kinetic complexity. Although the kinetic analysis may not be unique, it appears that the azide complex cannot be fully formed, as judged by spectrophotometric changes, even with an infinitely great [N3-]. This is interpreted as resulting from an equilibrium between the final spectroscopically observable azide complex and an intermediate species whose spectrum is not substantially different from that of MnIIIMb itself. The two forms of the azide complex appear to exhibit roughly equal proportions at 3 degrees C. We propose that this intermediate is a weak Mn3+ -azide complex in which the metal ion remains out-of-plane toward the imidazole of the proximal histidine, but that the metal lies toward the anion in the "final" complex.

Animals↗

PubMed2006

Azotobacter vinelandii vanadium nitrogenase: formaldehyde is a product of catalyzed HCN reduction, and excess ammonia arises directly from catalyzed azide reduction.

The Mo-nitrogenase-catalyzed reduction of both cyanide and azide results in the production of excess NH3, which is an amount of NH3 over and above that expected to be formed from the well-recognized reactions. Several suggestions about the possible sources of excess NH3 have been made, but previous attempts to characterize these reactions have met with either limited (or no) success or controversy. Because V-nitrogenase has a propensity to release partially reduced intermediates, e.g., N2H4 during N2 reduction, it was selected to probe the reduction of cyanide and azide. Sensitive assay procedures were developed and employed to monitor the production of either HCHO or CH3OH (its further two-electron-reduced product) from HCN. Like Mo-nitrogenase, V-nitrogenase suffered electron-flux inhibition by CN- (but was much less sensitive than Mo-nitrogenase), but unlike the case for Mo-nitrogenase, MgATP hydrolysis was also inhibited by CN-. V-Nitrogenase also released more of the four-electron-reduced intermediate, CH3NH2, than did Mo-nitrogenase. At high NaCN concentrations, V-nitrogenase directed a significant percentage of electron flux into excess NH3, and under these conditions, substantial amounts of HCHO, but no CH3OH, were detected for the first time. With azide, in contrast to the case for Mo-nitrogenase, both total electron flux and MgATP hydrolysis with V-nitrogenase were inhibited. V-Nitrogenase, unlike Mo-nitrogenase, showed no preference between the two-electron reduction to N2-plus-NH3 and the six-electron reduction to N2H4-plus-NH3. V-Nitrogenase formed more excess NH3, but reduction of the N2 produced by the two-electron reduction of N3(-) was not its source. Rather, it was formed directly by the eight-electron reduction of N3(-). Unlike Mo-nitrogenase, CO could not completely eliminate either cyanide or azide reduction by V-nitrogenase. CO did, however, eliminate the inhibition of both electron flux and MgATP hydrolysis by CN-, but not that caused by azide. These different responses to CO suggest different sites or modes of interaction for these two substrates with V-nitrogenase.

Ammonia↗

PubMed2003

RNA-tethered phenyl azide photocrosslinking via a short-lived indiscriminant electrophile.

Arylazide mediated photocrosslinking has been widely used to obtain structural constraints in biological systems, even though the reactive species generated upon photolysis in aqueous solution have not been well characterized. We establish a mechanistic framework for formation of adducts between photoactivated 3-hydroxyphenyl azide and RNA. Tethered to an internal site in an RNA duplex via a 2'-amido linkage, photolysis of the aryl azide yields a cross-strand cross-link. Analysis of the ability of reagents with diagnostic reactivities to intercept formation of this cross-strand cross-link supports the assignment that the photoactivated intermediate is the ketenimine or a ketenimine-derived ring expansion product. Neither the initially produced singlet nitrene nor the subsequently formed triplet nitrene contribute to cross-link formation. Argon matrix and time-resolved solution experiments show that photolysis of free 3-hydroxyphenyl azide releases (in <or=20 ns) either a ketenimine or azepinone intermediate that reacts with nucleophiles. Adenosine, uridine, and guanosine monophosphate nucleotides have approximately equivalent abilities to quench the cross-strand cross-link, indicating that the photoactivated intermediate reacts broadly with functional groups in RNA. The reactive intermediate forms an adduct with adenosine monophosphate when tethered to both an RNA duplex or unstructured single strand; thus, cross-link formation is independent of the local RNA environment. The lifetime of the reactive intermediate generated upon photolysis of free 3-hydroxyphenyl azide in 50 mM Hepes diamine buffer is found to be 60-160 micros or significantly shorter than large scale RNA folding events. RNA-tethered 3-hydroxyphenyl azide cross-linking in aqueous buffer can thus be used with confidence to map structural neighbors, including most dynamic interactions, in RNA.

Acrylamide↗

PubMed1998

Induction of GLUT1 mRNA in response to azide and inhibition of protein synthesis.

Incubation of Clone 9 cells, a nontransformed rat liver cell line, in the presence of 5 mM azide results in an induction of GLUT1 mRNA which becomes detectable after approximately 3 h of continuous exposure to the agent. In examining the role of on-going protein synthesis in this response, we found that: (i) the induction of GLUT1 mRNA by azide was not inhibited by anisomycin, (ii) exposure to anisomycin alone also resulted in increased GLUT1 mRNA content, and (iii) the increments in GLUT1 mRNA content in the presence of both azide and anisomycin were additive. Following exposure to 30 microM anisomycin, the increase in GLUT1 mRNA content became evident at 1 h, reached a maximum level of approximately 7-fold at 3 h, then slowly decreased but remained elevated at approximately 2-fold control levels at 12 h. Transcription of the GLUT1 gene, estimated by nuclear run-on assay, was stimulated 1.4 +/- 0.1 and 1.6 +/- 0.2-fold in cells exposed to anisomycin for 1 and 2 h, respectively (p < 0.05 for both). Upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with a half-life of 1.9 +/- 0.4 h in control cells, while in contrast, GLUT1 mRNA half-life was 4.6 +/- 0.8 h in cells exposed to anisomycin. The induction of GLUT1 mRNA by anisomycin was half-maximal at approximately 3 microM, whereas inhibition of leucine incorporation and stimulation of Stress Activated Protein Kinase (SAPK), measured as c-Jun N-terminal kinase activity, were half-maximal at approximately 0.3 and approximately 0.05 microM anisomycin, respectively. GLUT1 mRNA content was also increased by the protein synthesis inhibitor emetine, and the effect was associated with no stimulation of SAPK activity. Finally, SAPK activity was minimally stimulated in cells exposed to azide. It is concluded that: (1) on-going protein synthesis is not necessary for the induction of GLUT1 mRNA content in response to azide, (2) the induction of GLUT1 mRNA by anisomycin is related to its activity to inhibit protein synthesis, and (3) under basal conditions, a rapidly turning-over putative protein exerts a negative regulatory effect on GLUT1 mRNA expression.

Animals↗

PubMed1998

Optimisation of a reagentless laccase electrode for the detection of the inhibitor azide.

Here we report on the optimisation of a reagentless enzyme sensor for the detection of azide based on the mediated reduction of O2 by a laccase enzyme co-immobilised in a redox hydrogel on electrode surfaces. The sensor response is shown to be influenced by the enzyme loading, the electrolyte pH and ionic strength. The response of the sensor is stable, decreasing by only 25% over a sixteen-hour period. Reproducible inhibition curves for the determination of azide levels from cyclic voltammetric scans can be obtained by normalisation of the sensor response. The resulting enzyme inhibition biosensor can detect levels of azide as low as 2.5 microM under these conditions. Constant potential amperometric detection at the laccase enzyme electrode in a flow injection set-up yields a peak current for inhibition of the mediated reduction of O2. Reproducible peak currents and areas (8.0 and 6.3% RSD, respectively, for n = 11) are obtained for repeated injections of 100 microM azide. Reproducible response curves can be obtained by injection of a 25 mM azide sample and assuming that the peak height and peak area obtained represent 100% inhibition of the enzyme.

Biosensing Techniques↗

PubMed2005

Azide rearrangements in electron-deficient systems.

The azide group has a diverse and extensive role in organic chemistry, reflected in the power of azide anion as a strong nucleophile, the role of organic azides as excellent substrates for cycloaddition reactions, the uses of azides as precursors of amines and nitrenes, and azide rearrangements known as the Curtius and Schmidt reactions. In recent years the scope of the Schmidt reaction has begun to be explored in depth, so that it now represents an important reaction in synthetic chemistry. This tutorial review analyses and summarises key recent developments in the field of Schmidt reactions.

Alcohols↗

PubMed1998

pH-dependent inhibition by azide and fluoride of the iron superoxide dismutase from Propionibacterium shermanii.

The iron-containing superoxide dismutase from Propionibacterium shermanii shows, in contrast with other iron superoxide dismutases, only a minor inhibition by azide or fluoride (10-100 mM) of up to 23% at pH 7.8. The activity of the protein with Mn bound to the active site was not diminished under the same conditions. The binding constant between azide and the Fe3+ ion was determined as approx. 2 mM and for fluoride approx. 2.3 mM; they are so far comparable to those known for other iron superoxide dismutases. This seems to be a discrepancy because all other iron superoxide dismutases so far known are described as being inhibited by 50-70% by 10 mM azide. However, towards lower pH there was a drastically increased inhibition by both anions. At pH 6.8 about 80% inhibition was exhibited by azide or fluoride at a concentration of 10 mM or higher. In contrast, on increasing the pH, azide or fluoride still bound to the Fe3+ at the active site but their inhibition capacity decreased. This observation implies that both anions bind to the metal at a position that is empty at low pH, whereas at higher pH water or a negatively charged hydroxyl anion is bound. It is likely that the superoxide anion binds to the same position and has to replace the sixth ligand, leading to a diminished catalytic activity of the superoxide dismutase owing to steric and/or electrostatic inhibition of the ligand.

Azides↗

PubMed2006

Vibrational energy relaxation of azide in water.

Vibrational lifetimes of the asymmetric stretch fundamental of azide anion in normal and heavy water have been measured experimentally, with results in the range of a few picoseconds. This is an interesting problem for theoretical study because of the competition between intramolecular (relaxation to the other excited vibrational states of azide) and purely intermolecular (relaxation to azide's ground vibrational state) pathways. In addition it is important to understand the origin of the solvent isotope effect. Building on the seminal work of Morita and Kato [J. Chem. Phys. 109, 5511 (1998)], the authors develop a simple model based on a two-dimensional description of the azide stretching vibrations. A novel aspect of their theory is the use of an "on-the-fly" optimized quantum mechanical/molecular mechanical approach to calculate the system-bath coupling. Their theoretical lifetimes are in good agreement with experiment for azide in both normal and heavy water. They find that the predominant relaxation pathway is intramolecular. The solvent isotope effect arises from the different librational frequencies in normal and heavy water.

Azides↗

PubMed1995

Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Experimental evidence for proton transfer via a hydrogen-bonded network in a membrane protein is presented. Bacteriorhodopsin's proton transfer mechanism on the proton uptake pathway between Asp-96 and the Schiff base in the M-to-N transition was determined. The slowdown of this transfer by removal of the proton donor in the Asp-96-->Asn mutant can be accelerated again by addition of small weak acid anions such as azide. Fourier-transform infrared experiments show in the Asp-96-->Asn mutant a transient protonation of azide bound to the protein in the M-to-N transition and, due to the addition of azide, restoration of the IR continuum band changes as seen in wild-type bR during proton pumping. The continuum band changes indicate fast proton transfer on the uptake pathway in a hydrogen-bonded network for wild-type bR and the Asp-96-->Asn mutant with azide. Since azide is able to catalyze proton transfer steps also in several kinetically defective bR mutants and in other membrane proteins, our finding might point to a general element of proton transfer mechanisms in proteins.

Amino Acid Sequence↗

PubMed1998

Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F1-ATPase catalytic sites measured using specific tryptophan probes.

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F1-ATPase was measured in the presence of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in catalytic sites were used to determine nucleotide binding parameters. Azide brought about small decreases in Kd(MgATP) and Kd(MgADP). Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state with three catalytic site-bound MgATP and zero bound MgADP. These results rule out the idea that azide inhibits by "trapping" MgADP. Rather, azide blocks the step at which signal transmission between catalytic sites promotes multisite hydrolysis. Aurovertin bound with stoichiometry of 1.8 (mol/mol of F1) and allowed significant residual turnover. Cycling of the aurovertin-free beta-subunit catalytic site through three normal conformations was indicated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that it acts to slow the switch of catalytic site affinities ("binding change step") subsequent to MgATP hydrolysis. Dicyclohexylcarbodiimide shifted the ratio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affecting Kd(MgATP) values. Like azide, it also appears to affect activity at the step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolysis.

Adenosine Diphosphate↗

PubMed2001

Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.

The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.

Azides↗