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PubMed2004

Detection of cannabinoid CB1, adenosine A1, muscarinic acetylcholine, and GABA(B) receptor-dependent G protein activity in transducin-deactivated membranes and autoradiography sections of rat retina.

1. Several G-protein-coupled receptors (GPCRs) have been localized to various layers of the vertebrate retina, using autoradiographic and immunohistochemical techniques, but the functional data concerning G protein activation are limited. Here, we establish optimized assay conditions to detect receptor-dependent G protein activity in membranes and tissue sections of the rat retina. 2. Agonist-stimulated [35S]GTPgammaS-binding responses were characterized for the Gi/o-linked adenosine A1, cannabinoid CB1, m2/m4 muscarinic acetylcholine, and GABA(B) receptors. Initial assumption was that G protein activity under "basal conditions" is high due to enrichment and activity of rhodopsin and transducin in this tissue. 3. We found that pretreatment of retina membranes with hydroxylamine (10 mM), a rhodopsin-inactivating drug, substantially (up to 60%) reduced basal G protein activity, thereby improving signal-to-noise ratio to detect agonist-stimulated G protein activation for all studied receptors. [35S]GTPgammaS autoradiography revealed that hydroxylamine specifically reduced basal binding in the transducin-enriched photoreceptor layer. In contrast, hydroxylamine did not affect GPCR signaling in brain membranes, indicating specific action on retinal transducin. 4. For all studied receptors, [35S]GTPgammaS autoradiography allowed localization of G protein activity to different retinal layers, with the bulk of signal detected in the ganglion cell layer. Strongest responses were observed for adenosine and muscarinic receptor agonists. Additional G protein activity was detected in the inner plexiform layer. 5. Responses to all tested agonists were reversed in the presence of appropriate receptor-selective antagonists, indicating receptor-mediated G protein activation.

Adenosine↗
PubMed2005

Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical.

Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60 degrees rotational angle of C(alpha)-(C(beta)-C(1))-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens.

Amino Acid Sequence↗
PubMed1980

Covalent binding and hemolytic activity of complement proteins.

We report the inactivation of the third component of complement (C3) by hydroxylamine. C3 hemolytic and covalent binding activities decline with identical kinetics, demonstrating a direct correlation between the two activities. We conclude that covalent, surface-bound C3b is hemolytically active. The inactivation of C3 is first order with respect to hydroxylamine. We also studied C3 inactivation with [14C]methylamine. The inactivation corresponds quantitatively with the labeling of C3 in the C3d domain. The data obtained support the following hypothesis: there is an internal thioester within C3 which becomes highly reactive on activation to C3b, and C3b binds to receptive surfaces by transfer of the acyl function of the thioester to a hydroxyl group on the receptive surface. This proposed model for the reaction of C3 with receptive surfaces also applies to C4, which binds to membrane surfaces covalently and is able to be inactivated by hydroxylamine and methylamine. C5, on the other hand, is not inactivated by treatment with the amines.

Binding Sites↗
PubMed2000

Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase.

The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of gamma-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase.

Binding Sites↗
PubMed1999

Mechanisms underlying gastric antiulcerative activity of nitroxides in rats.

Reactive oxygen-derived species and redox-active metals are implicated in mediation of the pathogenesis of gastric mucosal damage and ulceration. Therefore, common strategies of intervention employ metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants (LMWA). The aim of the present study was to elaborate the mechanism(s) responsible for the protection provided by nitroxide radicals in the experimental model of gastric ulceration. Fasted male rats were treated ig with 1 ml 96% ethanol, with or without ig pretreatment with nitroxide or hydroxylamine. In several experiments, rats were injected ip or iv with iron(III) or iron(II) prior to ethanol administration. Rats were sacrificed 10 min after ethanol administration, the stomach was removed, washed and lesion area measured. Pretreatment with iron(III) complexed to nitrilotriacetate or citrate, aggravated the extent of the gastric injury. Conversely, iron(II) inhibited the formation of lesions. The nitroxides were rapidly reduced to their respective hydroxylamines and demonstrated antiulcerative activity for rats treated with iron. However, injecting the hydroxylamine resulted in a similar tissue distribution of nitroxide/hydroxylamnine but did not provide protection. The results show that: (a) the nitroxide radicals, rather than their respective non-radical reduced form, are the active species responsible for protection; (b) nitroxides protect by dismutating O2*- and possibly indirectly increasing the NO level; (c) unlike classical LMWA which are reducing agents, nitroxides inhibit gastric damage by acting as mild oxidants, oxidizing reduced metals and pre-empting the Fenton reaction; and (d) the nitroxides act catalytically as recycling antioxidants.

Animals↗
PubMed1976

Isolation, properties, and crystallization of an iron-chlorin protein from Aspergillus niger.

A green iron-chlorin protein was purified 160-fold from a lyophilized extract of Aspergillus niger by ion-exchange chromatography and gel filtration with a yield of 25%. The purified preparation appeared nearly homogeneous on sedimentation analysis and the sedimentation coefficient of the protein at infinite dilution was 13.4S. Its molecular weight was calculated to be 2.8--3.2 X 10(5) from sedimentation and gel filtration data. The ferric form of the protein had absorption maxima at 587.5 and 708 nm in the visible region and a Soret band at 404 nm. High-spin ESR signals of a rhombically distorted ferric iron-chlorin complex were observed at g = 6.5 and 5.3 together with unidentified, weaker signals at g = 4.3 and 2.0. The ferric form reacted readily with cyanide to give a complex showing absorption peaks at 422 and 632 nm and a shoulder at about 595 nm. When the protein combined with cyanide its high-spin ESR signals disappeared and low-spin signals at g = 1.88, 2.29, anous form having absorption maxima at 622 and about 410 nm. The rate of reduction by dithionite was slightly reduced by the presence of either nitrite or sulfite, and greatly accelerated by the presence of hydroxylamine. The reduced spectrum obtained in the presence of hydroxylamine had maxima at 620 and about 420 nm. The ferric cyanide complex did not show any spectral change on addition of dithionite. The green prosthetic group could be extracted with acidified acetone and the absorption maxima of the pyridine ferrihemochrome were at 413 and 599 nm. On removal of metal from the prosthetic group the characteristic spectrum of a chlorin was obtained. The absorption maxima of a solution of the chlorin in benzene were at 403, 501, 537, 576, 595, and 655 nm, the 655 nm band being strongest of those in the visible region. No significant amount of flavin was detected in the purified preparation. The iron-chlorin protein catalyzed methyl viologen-linked reduction of hydroxylamine and also that of nitrite at a slower rate under the same conditions, but not evidence that it reduced sulfite was obtained in the present study. The purified preparation also had high catalase [ec 1.11.1.6] activity. Crystalline material was obtained by gradual concentration of the purified preparation at about pH 6.

Aspergillus↗
PubMed2006

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances.

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.

Ammonia↗
PubMed1981

A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554.

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.

Ammonia↗
PubMed1982

Cytochrome c554 as a possible electron donor in the hydroxylation of ammonia and carbon monoxide in Nitrosomonas europaea.

Mechanism of ammonia oxidation was studied in the reconstituted system of Nitrosomonas membrane fraction plus the Nitrosomonas cytochrome c554. The cytochrome c554 was reduced by hydroxylamine, hydrazine, and ammonia and the reduced cytochrome was oxidized upon the addition of ammonia or carbon monoxide. The oxidation of carbon monoxide in the presence of hydroxylamine or hydrazine was studied as a possible assay method for ammonia hydroxylase where hydroxylamine or hydrazine was supplying the reducing power required for the hydroxylation of carbon monoxide. The stoichiometry of the reaction, Km values for substrates, and effects of pH and inhibitors were investigated. It is concluded that carbon monoxide, a competitive inhibitor for ammonia oxidation, is an alternate substrate for ammonia hydroxylase using the reduced cytochrome c554 as the reducing power.

Ammonia↗
PubMed2003

Acceleration of key reactions as a strategy to elucidate the rate-limiting chemistry underlying phototransduction inactivation.

PURPOSE: A reconstituted system was used to establish a strategy to determine the rate-limiting chemistry responsible for recovery of the dim-flash response in rod photoreceptors. METHODS: A general approach for identifying the rate-limiting step in a series of reactions is to evaluate the consequences of accelerating each step separately, while monitoring the rate of formation of the end product of the series. This strategy was applied to the reactions involved in quenching phototransduction in bovine rod outer segment (bROS) homogenates. The decay of photoactivated rhodopsin (R*) and inactivation of transducin by guanosine triphosphate (GTP) hydrolysis are the leading candidates for limiting the rate of phototransduction turn-off. These reactions were accelerated separately and together by adding hydroxylamine and/or the regulator of G-protein signaling-9 catalytic domain (RGS9d) while monitoring phosphodiesterase (PDE) activity triggered by a pulse of light in bROS homogenates. RESULTS: PDE activity in bROS homogenates triggered by a flash of light returned to its dark value with a rate constant of 0.087 +/- 0.002 seconds in this system. The rate of PDE recovery increased to 0.11 +/- 0.004 seconds when R* decay was accelerated with 10 to 50 mM hydroxylamine, suggesting that R* inactivation limits the rate of phototransduction turn-off under these conditions. Adding both hydroxylamine and RGS9d, a factor that accelerates transducin inactivation, increased the rate of PDE decay even further. RGS9d had no effect on PDE recovery kinetics unless quenching of R* was also accelerated. CONCLUSIONS: Under in vitro conditions in bROS homogenates, the quenching of R* normally limits the rate of phototransduction shut-off. If R* decay is accelerated, inactivation of transducin by GTP hydrolysis becomes rate limiting. This study offers a general approach that could be used to investigate the rate-limiting chemistry of phototransduction turn-off in vivo.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMedDate not supplied

Kinetic investigations of the autoxidation of adrenalin.

Generation rates of superoxide anions (O2-) by autoxidizing adrenalin at pH 9.5 were determined in solutions containing either superoxide dismutase (0.85 M-1 S-1) or hydroxylamine (0.0185 M-1 S-1) as competitive scavengers. The rate constants of O2- with adrenalin and hydroxylamine were calculated for neutral and alkaline solutions. The respective values were for adrenalin: 5.6 X 10(4) M-1 S-1, pH 7.8; 7.0 X 10(3) M-1 S-1, pH 9.5--and for hydroxylamine 5.9 X 10(4) M-1 S-1, pH 7.8; 3.4 X 10(4) M-1 S-1, pH 9.5. The effects of various competitors and O2- sources on the rate constants were compared.

Epinephrine↗
PubMed1998

Carbonic anhydrase inhibitors: inhibition of isozymes I, II and IV with N-hydroxysulfonamides--a novel class of intraocular pressure lowering agents.

A series of N-hydroxy sulfonamides has been prepared by reaction of alkyl-, arylalkyl- and arylsulfonyl halides or sulfonic acid anhydrides with hydroxylamine. Structurally related inhibitors were also obtained from acyl chlorides and hydroxylamine, as well as by reaction of tosyl isocyanate with hydroxylamine, sulfamic acid and sulfamide. Inhibition of three carbonic anhydrase (CA) isozymes, hCA I, hCA II and bCA IV (h = human; b = bovine) with the prepared compounds has been investigated. Good inhibitors, as well as compounds with moderate activity against these isozymes were detected, depending on the R group to which the SO2NHOH or CONHOH moieties were attached. Susceptibility to inhibition was generally: hCA II > bCA IV >> hCA I. Some of the new inhibitors showed very good antiglaucoma action when administered directly into the eye in experimental animals, acting as more efficient intraocular pressure lowering agents as compared to the clinical drug dorzolamide. This constitutes an encouraging result for obtaining novel antiglaucoma drugs from this class of CA inhibitors.

Acetamides↗
PubMed1990

[Measurement of superoxide radicals produced by human activated neutrophils by accumulation of stable nitroxide radicals detected by MR spectroscopy].

An important index of neutrophil function is the production of superoxide radicals (O2-) upon activation. Thus a development of a new adequate assay of O2- generation measurement is of great interest for phagocyte researchers. The present article considers the quantitative determination of O2- generation based on the interaction of O2- with 1-oxy-2,2,6,6-tetramethyl-4-oxypiperidine producing 4-oxo-2,2,6,6-piperidine-1-oxyl, detected by ESR. The kinetic curve of nitroxyl radical (NR) formation has a linear character. The NR formation rate after a short induction period (appr. 2 min.) approaches 3.3 X 10(-3) M/s, where cell concentration was 4 X 10(5) per ml. Hydroxylamine (3.8 mM) auto-oxidation rate is negligible as compared with activated neutrophils and is equal to 2 X 10(-9) M/s. Sensitivity NR to the presence of superoxide dismutase (SOD) came as evidence that NR formation is due O2- radicals. SOD (10(-7) M) inhibits NR formation by 90%. Hydroxylamine oxidation by O2- is an irreversible reaction--20-min incubation of activated neutrophils with NR do not influence NR concentration. The NR generation rate dependence upon the neutrophil concentration is linear in the cell concentration range from 4 X 10(5 up to 6 X 10(6) per ml. In this range a quantitative measurement of O2- production is suitable. The sensitivity of hydroxylamine assay is close to the sensitivity of chemiluminescent method, but specificity is higher, as SOD inhibits chemiluminescence only by 50%.

Free Radicals↗
PubMed1985

Neuropharmacological screening of newly-synthesized 2-substituted derivatives of the barbituric acid.

Neuropharmacological screening is applied of fourteen newly-synthesized 2-substituted derivatives of the barbituric acid (the first seven compounds contain a hydroxylamine group, the rest--a hydrazine group). The derivatives were studied using Irwin's screening test (1964), modified by Nikolova and Daleva (1968), after being introduced intraperitoneally in albino mice of both sexes. Excitatory phenomena prevail in the compounds containing the hydrazine group. The derivatives containing hydroxylamine group manifest predominantly inhibitory action of the central nervous system--hypnotic and myorelaxing activity. The intensity of the hypnotic action of the compounds is due not only to the hydroxylamine group at site 2, but also to the variation of the second substitute at site 5, with ethyl group as constant substitute. Considerable hypnotic effect is observed at the second substitute n-propyl group (HB-2) and its branching (sec. pentyl, HB-7). Two promising compounds for future more detailed studies--HB-7 and HB-2--become apparent.

Animals↗
PubMed1979

[Effectiveness of oral immunization of white mice with a complex S. typhimurium antigen].

The protective properties of hydroxylamine preparation obtained from a virulent strain of S. typhimurium were studied in experiments with natural infection after a single oral immunization. The new data obtained in these experiments suggest that the treatment of bacteria with hydroxylamine allows to produce the preparation which, when administered orally, has the immunizing dose only 20 times as great as its immunizing dose for subcutaneous administration. The action of gastric juice on hydroxylamine preparation, as well as the duration and specificity of immunity induced by the oral administration of this preparation were studied. The oral administration of some adjuvants was found to make it possible to considerably decrease the effective dose of the vaccine.

Animals↗
PubMed1976

Evidence for an ester bond between thrombin and heparin cofactor.

Heparin cofactor, a thrombin inhibitor, is purified from human plasma by affinity chromatography on heparin-agarose. The nature of the binding between thrombin and the inhibitor is studied by treatment of the complex with 6 M guanidinium chloride, hydroxylamine, and dilute alkali. The complex is not dissociated during gel chromatography in 6 M guanidinium chloride. This result supports an earlier proposal that formation of the complex includes the formation of a covalent bond. Treatment of dodecyl sulfate-denatured complex with hydroxylamine results in dissociation of the complex to yield free thrombin and heparin cofactor. Hydroxylamine does not dissociate the complex unless it is denatured. The complex is also dissociated in dilute sodium hydroxide (pH 12) solutions. These results indicate that the covalent bond between thrombin and the inhibitor is a carboxylic ester.

Alkalies↗
PubMedDate not supplied

[Molecular mechanisms of receptor-potential generation by the photoreceptor. III. Conformational transition responsible for the tail end of the photoresponse of an artificial lipid membrane modified by fragments of the external segments of rods].

The kinetics of photoinduced changes of protein fluorescence of cattle visual pigment was studied in the presence of hydroxylamine. The rate constant of fluorescence increase is proportional to NH2OH concentration when it is less than 0.4 M. It reaches the maximal magnitude (3.3 +/- 1 sec-1) at higher hydroxylamine concentration. Fluorescence increase rate is controlled by the rate of chemical reaction of rhodopsin with hydroxylamine. It is limited by conformational rearrangement of opsin. This rearrangement does not induce absorbance spectrum change of visual pigment, but confers to it the capability to react with NH2OH and NaBH4. Kinetic parameters of this rearrangement (tau 20 degrees C approximately 300 msec, Eact = 19 +/- 2 kcal/mole) coincide with kinetic parameters of diminishing of the photoresponse of artificial lipid membrane modified by fragments of rod outer segments in the temperature range studied (+2 divided by +25 degrees C).

Animals↗
PubMed1982

Further studies of the suicide inactivation of purified rat liver cytochrome P-450 by chloramphenicol.

The kinetics and reversibility of the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol have been investigated with the use of a reconstituted monooxygenase system purified from liver microsomes of phenobarbital-treated rats. At a ratio of 1 unit of NADPH-cytochrome P-450 reductase per nanomole of cytochrome P-450 and a chloramphenicol concentration of 1 mM, the t1/2 for the inactivation of cytochrome P-450 is less than 2 min. The inactivated cytochrome regains some of its activity upon incubation at 25 degrees or 37 degrees, and experiments with [14C]chloramphenicol show that this partial reactivation is accompanied by the release of some of the 14C originally bound covalently to the cytochrome P-450. Previous work has shown that the 14C-labeled material spontaneously released from 14C-labeled cytochrome P-450 is in the form of oxalic acid, and that the latter is derived from a hydroxylamine-labile adduct of chloramphenicol and cytochrome P-450 [Biochem. Pharmacol. 30:875-881 (1981)]. In the present investigation the 14C-labeled material released by hydroxylamine was identified as the hydroxamic acid of oxalic acid. Trapping experiments with the amino acid cysteine suggest that the adduct, the spontaneous degradation of which appears to be involved in the reactivation of cytochrome P-450, contains an ester rather than a thioester linkage between cytochrome P-450 and a metabolite of chloramphenicol. However, this metabolite may not be identical with chloramphenicol oxamyl chloride, which was the active metabolite implicated in the formation of the 50% covalently bound material which was stable to hydroxylamine treatment.

Animals↗