Absence Of Hydroxylamine Research Articles

Hydroxylamine promoted Fe(III) reduction in H2O2/soil systems for phenol degradation.

Production of hydroxyl radicals (•OH) upon the oxidation of solid Fe(II) by O2 or H2O2 in soils and sediments has been confirmed, which benefits in situ remediation of contaminants. However, Fe(III) reduction by H2O2 is rate-limiting. Accelerating the Fe(III)/Fe(II) cycle could improve the efficiency of remediation. This study intended to use hydroxylamine to promote Fe(III)/Fe(II) cycle during 100g/L soil oxidation by H2O2 for phenol degradation. The removal of phenol was 76% in 3h during soil oxidation with 1mM H2O2 in the presence of 1mM hydroxylamine but was negligible in the absence of hydroxylamine. Fe(III) in the soil was reduced to 0.21mM Fe(II) by 1mM hydroxylamine in 30min. The accelerated cycle of Fe(III)/Fe(II) in the soil by hydroxylamine could effectively decompose H2O2 to produced •OH, which was responsible for the effective enhancement of phenol degradation during soil oxidation. Under the conditions of 1mM H2O2 and 100g/L soil, the pseudo-first-order kinetic constant of phenol degradation increased proportionally from 0.0453 to 0.0844min-1 with the increase of hydroxylamine concentrations from 0.5 to 1mM. The kinetic constant also increased from 0.0041 to 0.0111min-1 with H2O2 concentration increased from 0.5 to 2mM, while it decreased from 0.0100 to 0.0051min-1 with soil dosage increased from 20 to 200g/L. In addition, column experiments showed that phenol (10mg/L) degradation ratio kept at about 48.7% with feeding 2mM hydroxylamine and 2mM H2O2 at 0.025 PV/min. Column experiments suggested an optional application of hydroxylamine and H2O2 for in situ remediation. The output of this study provides guidance and optional strategies to enhance contaminant degradation during soil oxidation.

FTIR Study of the Photoreaction of Bovine Rhodopsin in the Presence of Hydroxylamine

In bovine rhodopsin, 11-cis-retinal forms a Schiff base linkage with Lys296. The Schiff base is not reactive to hydroxylamine in the dark, which is consistent with the well-protected retinal binding site. In contrast, under illumination it easily forms all-trans retinal oxime, resulting in the loss of color. This suggests that activation of rhodopsin creates a specific reaction channel for hydroxylamine or loosens the chromophore binding pocket. In the present study, to extract structural information on the Schiff base vicinity and to understand the changes upon activation of rhodopsin, we compared light-induced FTIR difference spectra of bovine rhodopsin in the presence and absence of hydroxylamine under physiological pH (approximately 7). Although the previous FTIR study did not observe the complex formation between rhodopsin and G-protein transducin in hydrated films, the present study clearly shows that hydrated films can be used for studies of the interaction between rhodopsin and hydroxylamine. Hydroxylamine does not react with the Schiff base of Meta-I intermediate trapped at 240 K, possibly because of decreased conformational motions under the frozen environment, while FTIR spectroscopy showed that hydroxylamine affects the hydrogen bonds of the Schiff base and water molecules in Meta-I. In contrast, formation of the retinal oxime was clearly observed at 280 K, the characteristic temperature of Meta-II accumulation in the absence of hydroxylamine, and time-dependent formation of retinal oxime was observed from Meta-II at 265 K as well. The obtained difference FTIR spectra of retinal oxime and opsin are different from that of Meta-II. It is likely that the antiparallel beta-sheet constituting a part of the retinal binding pocket at the extracellular surface is structurally disrupted in the presence of hydroxylamine, which allows the hydrolysis of the Schiff base into retinal oxime.

The Psb27 Protein Facilitates Manganese Cluster Assembly in Photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

Characterization of cis-Autoproteolysis of Polycystin-1, the Product of Human Polycystic Kidney Disease 1 Gene

Polycystin-1 (PC1), the PKD1 gene product, plays a critical role in renal tubule diameter control and disruption of its function causes cyst formation in human autosomal dominant polycystic kidney disease. Recent evidence shows that PC1 undergoes cleavage at the juxtamembrane G protein-coupled receptor proteolytic site (GPS), a process likely to be essential for its biological activity. Here we further characterized the proteolytic cleavage of PC1 at the GPS domain. We determined the actual cleavage site to be between leucine and threonine of the tripeptide HLT(3049) of human PC1. Cleavage occurs in the early intracellular secretory pathway and requires initial N-glycan attachment but not its subsequent trimming. We provide evidence that the cleavage occurs via a cis-autoproteolytic mechanism involving an ester intermediate as shown for Ntn hydrolases and EMR2.

Competition between ammonia derived from internal glutamine hydrolysis and hydroxylamine present in the solution for incorporation into UTP as catalysed by Lactococcus lactis CTP synthase

CTP synthase catalyses the reaction: glutamine + UTP + ATP → glutamate + CTP + ADP + P i. The reaction is greatly stimulated by the allosteric binding of GTP. In addition to glutamine that is hydrolysed by the enzyme to ammonia and glutamate, CTP synthase will also utilise external sources of amino donors such as NH 4Cl. This reaction is no longer dependent on allosteric activation by GTP. Hydroxylamine is also a substrate for Lactococcus lactis CTP synthase and results in the formation of N 4-OH CTP. This product has the feature that it absorbs at 300 nm where CTP absorption was shown to be greatly reduced and enabled the determination of N 4-OH CTP formation in the presence of CTP synthesis derived from glutamine hydrolysis. Differences in initial rates determined for the hydroxylamine dependent reaction at 291 nm in the presence and absence of glutamine and GTP were ascribed to simultaneous CTP and N 4-OH CTP synthesis in the presence of these compounds. A characterisation of the apparent inhibition by GTP and glutamine of N 4-OH CTP synthesis determined at 300 nm showed that glutamine dependent CTP synthesis occurs at a rate of about 60% of that in the absence of hydroxylamine. GTP dependent inhibition of the ammonium chloride dependent reaction of L. lactis CTP synthase by the glutamine analog glutamate γ-semialdehyde showed a partial inhibition with a maximum inhibition of about 60%. These results are interpreted in terms of a “half of the sites” mechanism for glutamine hydrolysis on CTP synthase.

A time-resolved FTIR difference study of the plastoquinone QA and redox-active tyrosine YZ interactions in photosystem II.

In this paper, we present the first time-dependent measurements of flash-induced infrared difference spectra of photosystem II (PSII) using Fourier transform infrared (FTIR) spectroscopy. With this experimental approach, we were able to obtain the YZoxQA-/YZQA vibrational difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 +/- 2 degrees C), with a spectral resolution of 4 cm-1 and a temporal resolution of 50 ms. In order to determine the dominant species in the FTIR spectrum at a particular point in time after an excitation flash, the decay kinetics of YZox and QA- were independently monitored by EPR and chlorophyll a fluorescence, respectively, under the same experimental conditions. These measurements confirmed that the addition of DCMU to Tris-washed PSII samples does not significantly affect the YZox decay, but does substantially slow down the QA- decay. By making use of the difference in the decay kinetics using DCMU, the QA-/QA signals could be separated from the YZox/YZ signals and a pure QA-/QA difference spectrum obtained. By comparison of the YZoxQA-/YZQA difference spectrum with the pure QA-/QA difference spectrum, a large differential band at 1706/1699 cm-1 could be identified and associated with YZ oxidation. In contrast, an intense band at 1478 cm-1, whose DCMU-sensitive decay follows the QA- decay based on the chlorophyll a fluorescence measurements, was present in all of the time-resolved spectra. Since no significant reversible Chl+ radicals could be detected by the EPR measurements under our experimental conditions, we confirm that this band most likely arises only from the semiquinone anion QA- [Berthomieu, C., Nabedryk, E., Mäntele, W., & Breton, J. (1990) FEBS Lett. 269, 363-367].

Immunosuppressive activities in the seminal plasma of infertile men: relationship to sperm antibodies and autoimmunity.

Semen samples from infertile men were assessed for sperm autoimmunity by direct immunobead assay for immunoglobulin (Ig)A and IgG sperm antibodies and mucus penetration test. Immunosuppressive activity in seminal plasma was measured by an in-vitro bioassay employing dose-dependent inhibition of phytohaemagglutinin-induced activation of rat thymocytes, in the presence or absence of hydroxylamine (0.1 mM), an inhibitor of polyamine oxidation. All seminal plasma samples, regardless of autoimmune status, caused inhibition of T-lymphocyte activation, and hydroxylamine reduced this bioactivity by appproximately 50%. Dialysis (<3500 molecular weight) also significantly reduced seminal plasma bioactivity, both in the presence and absence of hydroxylamine. In the presence of hydroxylamine, there was a negative correlation between IgA, but not IgG, antibody concentrations and lymphosuppressive activity in seminal plasma. Antibody-positive samples displaying impaired sperm function, as indicated by the mucus penetration test, had reduced activity compared with other samples. In contrast, there was no relationship between sperm autoimmunity and lymphosuppressive activity assayed in the absence of hydroxylamine. The data indicate that T-lymphocyte inhibition by human seminal plasma is due to multiple factors, and reduced amounts of these factors may contribute to the development and/or persistence of sperm autoimmunity in infertile men; however, differences in polyamine substrates available for oxidation in semen do not appear to be a major contributing factor.

Enzymatic production of L-tryptophan from DL-5-indolymethylhydantoin. Part II Enzymatic production of L-tryptophan from DL-5-indolylmethylhydantoin by mutants of Flavobacterium sp. T-523.

A bacterium isolated from soil, T-523, which hydrolyzes Dl-5-indolylmethylhydantoin to l- tryptophan, was classified as a bacterium belonging to the'genus, Flavobacterium. The prevention of tryptophan degradation by Flavobacterium sp. AJ-3912 (T-523) was investigated for improvement of the l-tryptophan production, because the l-tryptophan accumulated on enzymatic production from Dl-5-indolylmethylhydantoin was partially degraded. l-Tryptophan production by Flavo-bacterium sp. AJ-3912 increased with the addition of agents that inhibit tryptophan oxygenase, such as hydroxylamine and phenylhydrazine, to the reaction mixture. In the presence of an appropriate concentration of hydroxylamine, the amount of l-tryptophan produced from l-5- indolylmethylhydantoin reached 8.8 mg/ml, with a molar yield of 99% (in the absence of hydroxylamine, 7.1 mg/ml with a molar yield of 80%).The l-tryptophan productivity was also improved with the use of mutants with a genetically blocked tryptophan degradation pathway. In the p...

Effect of pyridoxine deficiency on aromatic L-amino acid decarboxylase in adult rat brain.

We have investigated the effect of pyridoxine deficiency on aromatic L-amino acid decarboxylase (AADC) using both dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5HTP) as substrates in the rat brain. The activity ratios of DOPA decarboxylase/5HTP decarboxylase measured under optimal substrate and cofactor concentrations were different in the cerebellum, cerebral cortex, corpus striatum and hypothalamus of the normal rat. In pyridoxine deficiency, there were no parallel decreases in DOPA and 5HTP decarboxylase activities in various brain regions. Dialysis of brain homogenates, in the presence and absence of hydroxylamine, resulted in a total or near total loss of 5HTP decarboxylase activity compared to DOPA decarboxylase activity, indicating that pyridoxal phosphate may be more tightly bound to DOPA decarboxylase than to 5HTP decarboxylase. These results, indicating that pyridoxine deficiency has differential effects on the activity of AADC, are consistent with our earlier observation of non-parallel changes in dopamine and serotonin content in various brain regions of the pyridoxine-deficient rat.

INTERACTION OF HYDROXYLAMINE WITH THE WATER‐OXIDIZING ENZYME INVESTIGATED VIA PROTON RELEASE*

Abstract— Photosynthetic water oxidation is a four‐step redox process which is driven by a one‐quantum‐one‐electron reaction center. Stepwise electron Abstract—ion from the water‐oxidizing enzyme is accompanied by stepwise proton release with the following stoichiometric pattern at given half‐rise times: 1 H+ (250 μs, S0→ S1):0 H+(S1→ S2): 1 H+ (200 μs, S2→ S3): 2 H+ (1.2 ms, S3→ S4→ S0). (Förster and Junge, 1985, preceding article in this issue). Hydroxylamine at low concentrations (≲100 μ M) appears to compete with water at the active site of the water‐ oxidizing enzyme. Its interference shifts the dark state of the water‐oxidizing enzyme by two steps backwards (Bouges, 1971). We found that the hydroxylamine‐induced shift was also reflected in the stoichiometric pattern and in the kinetics of proton‐release. In the presence of hydroxylamine, two protons per reaction center were released with a half‐rise time of ≅ 2 ms upon the first exciting flash given to dark adapted thylakoids. This was slower than observed for each of the protons released during unperturbed water oxidation. One proton was released upon the second flash. The half‐rise time of the main component observed upon the second flash in hydroxylamine‐treated samples agreed with the one observed upon the fourth flash in the absence of hydroxylamine, which had been attributed to the S0→ S1 transition. The two protons which were observed upon the first flash in hydroxylamine‐treated thylakoids may be due to hydroxylamine oxidation or to the association of water to the catalytic manganese center after detachment of oxidized hydroxylamine from its binding site.

Interplay between hydroxylamine, metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes

The decay reactions of metarhodopsin II and the dissociation of the complex between rhodopsin (in the metarhodopsin II state) and the GTP-binding protein (G-protein) (in its inactive, GDP-binding form) have been compared at various concentrations of hydroxylamine. The reactions of the chromophore were measured by absorption changes in the visible range, the complex dissociation by changes in the near-in-frared scattering. An additional monitor of the complex was given by the G-protein-dependent equilibrium between metarhodopsin I and metarhodopsin II. For all measurements, fragments of isolated bovine rod outer segments in suspension were used. In the absence of hydroxylamine, the rhodopsin-G-protein complex dissociated within 20–30 min at room temperature. The presence of hydroxylamine greatly accelerated (e.g., 5-fold at 1 mM NH 2OH) the dissociation. Under all conditions, the free, dissociated G-protein can reassociate to metarhodopsin II produced by subsequent bleaching. Dissociation of the metarhodopsin II-G-protein complex required the decay of photoproducts with a maximal absorbance of 380 nm, but was not affected by the simultaneous presence of metarhodopsin III or metarhodopsin III — like photoproducts with a maximal absorbance between 450 and 470 nm. Despite the acceleration of metarhodopsin II-G-protein dissociation by NH 2OH, metarhodopsin II-G-protein was relatively stabilized as compared to free metarhodopsin II. The ratio of the decay rates of free metarhodopsin II and metarhodopsin III-G-protein was increased as much as 10-fold in the presence of 25 mM NH 2OH. The results indicate a mutual interdependence of retinal, opsin and G-protein.

Acetylglutamate kinase. A mitochondrial feedback-sensitive enzyme of arginine biosynthesis in Neurospora crassa.

The radioisotopic method used to assay acetylglutamate kinase (EC 2.7.2.8) of Neurospora crassa has been shown to detect two distinct enzymatically catalyzed reactions. The enzymes were separated by differential centrifugation into a cytosolic activity and an organellar activity. Both activities required ATP and were thermal-labile. The cytosolic activity was insensitive to inhibition by arginine and formed a stable reaction product in the absence of hydroxylamine. The organellar activity had an absolute requirement for hydroxylamine in order to form a stable reaction product. The product of the cytosolic activity was separated from acetylglutamate hydroxamate (the product of the organellar activity) and was identified as the cyclic amide pyroglutamate by cation exchange chromatography. The organellar activity has been implicated in arginine biosynthesis by the following criteria: it was completely and specifically inhibited by arginine concentrations as low as 200 microM; its level was elevated 2-fold in a mutant strain with derepressed levels of arginine biosynthetic enzymes; and it was absent in an arginine auxotrophic strain (the cytosolic activity was present). The organellar activity co-sedimented with mitochondria during isopycnic gradient centrifugation. The metabolic problems posed by a mitochondrial location of a feedback-sensitive enzyme and the cytosolic location of its effector are discussed.

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.

Hydroxamic Acids IV: Hydroxide Effect in Hydroxylaminolysis of Ethyl Acetate

The kinetics and yields of acetohydroxamic acid from ethyl acetate were studied in order to assess the ability of excess hydroxide to decrease the sensitivity of the hydroxamic acid assay of a simple ester. Apparent first-order rate constants for hydroxylaminolysis of ethyl acetate were calculated from pH-stat data in the pH range 9.5–11.0 in the presence of 0.2–0.75 M hydroxylamine at 40°. First-order rate constants for ethyla cetate hydrolysis in the absence of hydroxylamine were determined under similar experimental conditions. The percent yields of acetohydroxamic acid were calculated as a function of pH and hydroxylamine concentration using the colorimetric assay of the hydroxamic acid-iron complex at 515 nm. Excess hydroxide was shown to increase the rate of reaction without decreasing hydroxamic acid yields. Based on these data, a rapid assay for ethyl acetate, acetylcholine, methacholine, and pilocarpine was developed, with high yields of the corresponding hydroxamic acids. Proposed mechanisms for participation by hydroxide ion in hydroxamic acid formation from simple esters are discussed.

Mechanism of Action of Guinea Pig Liver Transglutaminase: V. THE HYDROLYSIS REACTION

Abstract The transglutaminase-catalyzed hydrolysis of p-nitrophenyl acetate is effectively inhibited, in the absence of nucleophilic amine, by another substrate for the enzyme, benzyloxycarbonyl- (Z)-l-glutaminylglycine. Kinetic evaluation of this inhibition, with consideration of the dependence on calcium ion concentration and of previous studies of the amine transfer reaction, indicates that glutamine substrate is reversibly bound to different calcium-activated forms of the enzyme in the presence and in the absence of amine. The Ki value for Z-l-glutaminylglycine as an inhibitor of the esterolysis reaction was estimated to be 1.92 ± 0.5 x 10-3 m at pH 7.0. Examination of the calcium-dependent transglutaminase-catalyzed hydrolysis of Z-l-glutaminylglycine at two pH levels showed that the mechanism of hydrolysis conforms to that suggested by the esterolysis inhibition studies. The calcium ion requirements for hydrolysis were found to be very different from those for the amine transfer reaction. The Ka values of calcium in the hydrolysis reaction, estimated as 0.6 x 10-3 m at pH 7.0 and as 2.5 x 10-3 m at pH 5.4, are significantly lower than the Ka values of calcium reported for the amine transfer reaction and the Kd values derived from calcium-induced conformational changes in the enzyme protein. Agreement of the Km values for Z-l-glutaminylglycine at two pH levels in the hydrolysis reaction (approximately 2 x 10-3 m) with the Ki value for this Z-dipeptide as an inhibitor of esterolysis is consistent with the assumption that the hydrolysis reaction proceeds by an equilibrium mechanism. The effects of glutamine substrate on the rate of reaction between iodoacetamide and an essential sulfhydryl group of calcium-activated transglutaminase were investigated. The second order rate constant for reaction of the inactivator with this essential —SH group was found to be 96 x 103 liters mole-1 min-1, either in the presence or in the absence of hydroxylamine. Studies of the calcium ion requirement for this inactivation indicated that only the form of the enzyme in which metal ion is bound at both metal-binding positions reacts with iodoacetamide under the experimental conditions used. The decrease in reactivity of the essential —SH group resulting from Z-l-glutaminylglycine binding was found to be very different in the absence and in the presence of hydroxylamine. In the absence of amine the rate was decreased to about one-half that observed without substrate, whereas in the presence of amine the essential group appeared to be protected from reaction with iodoacetamide. These manifest differences, which are a function of steric changes induced by substrate binding, are in accord with the kinetic evidence for different mechanisms of substrate binding in the hydrolysis and amine transfer reactions.

Formation of hydroxamates during proteolysis by an enzyme system from yeast.

DURING re-investigation of an enzyme system, from yeast previously reported to form ‘activated’ peptides1 extensive proteolysis was noted and enzyme-catalysed hydroxylaminolysis of protein was suspected as the source of hydroxamate formation2. Acyl-enzyme complexes were visualized as intermediates rather than some form of ‘activated’ peptide originally suggested by Cooper et al.1. The main evidence for the latter hypothesis had been the appearance, after paper electrophoresis of the products of the reaction in the absence of hydroxylamine, of a band which gave a positive reaction to the hydroxylamine ferric chloride test. This test, however, may be misleading when conducted on paper3. The formation of hydroxamates from protein substrates by other proteolytic enzymes was also demonstrated2 and is presumably analogous to the well-known transamidation reactions recorded by many authors4–8. Such reactions have only been unequivocally demonstrated using simple substrates but have been invoked to explain the formation of plasteins9,10 and energy-independent incorporation of amino-acids into mitochondrial protein11.