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Frontiers in Immunology 2018Amylase trypsin inhibitors (ATI) can be found in all gluten containing cereals and are, therefore, ingredient of basic foods like bread or pasta. In the gut ATI can...
Amylase trypsin inhibitors (ATI) can be found in all gluten containing cereals and are, therefore, ingredient of basic foods like bread or pasta. In the gut ATI can mediate innate immunity via activation of the Toll-like receptor 4 (TLR4) on immune cells residing in the lamina propria, promoting intestinal, as well as extra-intestinal, inflammation. Inflammatory conditions can induce formation of peroxynitrite (ONOO) and, thereby, endogenous protein nitration in the body. Moreover, air pollutants like ozone (O) and nitrogen dioxide (NO) can cause exogenous protein nitration in the environment. Both reaction pathways may lead to the nitration of ATI. To investigate if and how nitration modulates the immunostimulatory properties of ATI, they were chemically modified by three different methods simulating endogenous and exogenous protein nitration and tested . Here we show that ATI nitration was achieved by all three methods and lead to increased immune reactions. We found that ATI nitrated by tetranitromethane (TNM) or ONOO lead to a significantly enhanced TLR4 activation. Furthermore, in human primary immune cells, TNM nitrated ATI induced a significantly higher T cell proliferation and release of Th1 and Th2 cytokines compared to unmodified ATI. Our findings implicate a causative chain between nitration, enhanced TLR4 stimulation, and adaptive immune responses, providing major implications for public health, as nitrated ATI may strongly promote inhalative wheat allergies (baker's asthma), non-celiac wheat sensitivity (NCWS), other allergies, and autoimmune diseases. This underlines the importance of future work analyzing the relationship between endo- and exogenous protein nitration, and the rise in incidence of ATI-related and other food hypersensitivities.
Topics: Adaptive Immunity; Amylases; Biomarkers; Cell Line, Tumor; Cell Survival; Cytokines; Dendritic Cells; Humans; Immunity, Innate; Immunophenotyping; Macrophages; Plant Proteins; T-Lymphocyte Subsets; Toll-Like Receptor 4; Triticum; Trypsin Inhibitors
PubMed: 30740114
DOI: 10.3389/fimmu.2018.03174 -
BMC Pharmacology Oct 2004Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an...
BACKGROUND
Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an important cellular signaling molecule in biological systems. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. However, data on the direct effect of reactive oxygen/nitrogen species (ROS/RNS) on HO function is incomplete. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite (ONOO-) on the in vitro catalytic activity of rat spleen (HO-1) and brain (HO-2) microsomal heme oxygenases.
RESULTS
Exposure to ONOO- led to concentration-dependent but reversible decreases in the activity of microsomal rat spleen and brain HO activity. Spleen HO activity was 100-fold more sensitive to ONOO--dependent inactivation compared to that of the brain, with IC50 values of 0.015 +/- 0.005 mM and 1.25 +/- 0.25 mM respectively. Inhibition of both rat spleen and brain microsomal HO activity was also observed with tetra-nitromethane, a tyrosine nitrating agent, as well as two NO donors, S-nitrosoglutathione (GSNO) and diethylamine NONOate (DEA-NONOate). However, no additive effect was found following the application of NO donors and ONOO- together.
CONCLUSION
These results indicate that ONOO- may regulate HO-1 and HO-2 activities by mechanisms that involve different interactions with these proteins. It is suggested that while nitration of tyrosine residues and oxidation of sulfhydryl groups may be involved, consideration should be given to other facets of ONOO- chemistry. This inhibition of HO activity offers a mechanism for cross talk between the nitric oxide synthase and HO systems.
Topics: Animals; Carbon Monoxide; Dose-Response Relationship, Drug; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; In Vitro Techniques; Male; Microsomes; Nitric Oxide; Nitrites; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Sulfhydryl Compounds
PubMed: 15498099
DOI: 10.1186/1471-2210-4-26 -
The Journal of Biological Chemistry Jan 1976Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of...
Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of tyrosyl peptides. Tyrosyl residues in the beta subunit (beta37, beta59) did not react with tetranitromethane in the intact hormone, but were nitrated in the isolated subunit. The sequence and extent of reaction of tetranitromethane with the tyrosyl residues in the alpha subunit was alpha21 = alpha92 = alpha93 (in intact hormone or isolated subunit) greater than alpha 41 (reacted in isolated subunit only) greater than alpha 30 (reacted in isolated subunit in 8 M urea only). Polymerization was observed as a side reaction in agreement with previous studies. The degree of polymerization appeared to be related to both primary sequence and tertiary structure, and for lutropin had the relation: alpha subunit (93% polymerized) greater than intact hormone greater than beta subunit (less than 40%). Polymerization observed with vasopressin was significantly greater than with oxytocin; for these peptides the tyrosine residues in the monomeric product were converted to 3-nitrotyrosine. Neither 3-nitrotyrosine nor tyrosine was detected in the polymerized by-products. In the tetranitromethane reaction with intact ovine lutropin, other reaction products charcterized by absorption spectra were found. Peptides isolated from these products lacked the characteristic 428 nm abosrption maxima of 3-nitrotyrosyl peptides and showed instead absorption in the 310 to 350 nm region. Similar products from tetranitromethane reactions with di- and tripeptides containing tyrosine have been observed previously (Boyd, N.D., and Smith, D.B. (1971) Can. J. Biochem, 49, 154-161), but they have not been studied in proteins. A possible relationship to the polymerization side reaction is suggested.
Topics: Amino Acid Sequence; Amino Acids; Animals; Binding Sites; Cattle; Kinetics; Luteinizing Hormone; Macromolecular Substances; Methane; Oxytocin; Peptide Fragments; Protein Binding; Protein Conformation; Sheep; Spectrophotometry; Spectrophotometry, Ultraviolet; Swine; Tetranitromethane; Vasopressins
PubMed: 1245474
DOI: No ID Found -
Journal of Lipid Research Oct 2002In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified...
In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO(x) species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO(x) species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.
Topics: Animals; Aorta, Thoracic; Blotting, Western; Culture Techniques; Endarterectomy, Carotid; Humans; Male; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitrogen Oxides; Precipitin Tests; Prostaglandin-Endoperoxide Synthases; Rats; Seminal Vesicles; Sheep; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry; Tyrosine
PubMed: 12364556
DOI: 10.1194/jlr.m200199-jlr200 -
The Journal of Biological Chemistry May 1981Repressor protein modified with N-ethylmaleimide has been used to determine the exclusive effects of tyrosine nitration by tetranitromethane. Since modification of...
Repressor protein modified with N-ethylmaleimide has been used to determine the exclusive effects of tyrosine nitration by tetranitromethane. Since modification of proteins with tetranitromethane generally results in both cysteine oxidation and tyrosine nitration, N-ethylmaleimide has been used to protect the cysteines in the repressor against oxidation in subsequent tetranitromethane reactions. Nitration of tyrosine residues in repressor previously reacted with N-ethylmaleimide results in loss of both specific and nonspecific DNA-binding activities. Na2S2O4 reduction of tetranitromethane-modified protein restores partial operator DNA-binding and complete nonspecific DNA-binding capability. Residues primarily affected are tyrosines 7 and 17, which are both in the NH2 terminus. Inter- and intramolecular cross-links which are observed in the modified protein can be minimized by altering reaction conditions; the cross-links present occur between sites located in the NH2 termini. Modification of the core protein also results in loss of the operator DNA-binding capacity, and subsequent reduction restores partial operator-binding activity. Both operator and nonspecific DNA-binding capabilities of the repressor protein are protected by the presence of nonspecific DNA during the tetranitromethane modification, and simultaneously the extent of nitration is decreased.
Topics: Binding Sites; DNA; Escherichia coli; Ethylmaleimide; Kinetics; Macromolecular Substances; Methane; Protein Binding; Repressor Proteins; Tetranitromethane; Transcription Factors; Tyrosine
PubMed: 7014561
DOI: No ID Found -
Journal of Bacteriology Nov 1985ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit... (Comparative Study)
Comparative Study
Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).
ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.
Topics: Amino Acids; Cross Reactions; Dithionitrobenzoic Acid; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Nucleotidyltransferases; Penicillium; Phenylglyoxal; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Species Specificity; Sulfate Adenylyltransferase; Temperature; Tetranitromethane; Urea
PubMed: 2997125
DOI: 10.1128/jb.164.2.674-683.1985 -
The Biochemical Journal Aug 1982The possible role of superoxide anion in 2-oxoglutarate-coupled dioxygenase reactions has been investigated. gamma-Butyrobetaine hydroxylase (EC 1.14.11.1) was inhibited...
The possible role of superoxide anion in 2-oxoglutarate-coupled dioxygenase reactions has been investigated. gamma-Butyrobetaine hydroxylase (EC 1.14.11.1) was inhibited by human erythrocyte superoxide dismutase (EC 1.15.1.1), probably due to release of Cu(2+) or Zn(2+), as the inhibition was more pronounced after heat-inactivation of the dismutase and as Cu(2+) was a potent inhibitor. Bovine superoxide dismutase and the Mn(2+)-containing superoxide dismutase from Escherichia coli were not inhibitory. Superoxide anion generated from xanthine/xanthine oxidase was not stimulatory and could not replace ascorbate. Thymine 7-hydroxylase (EC 1.14.11.6) and thymidine 2'-hydroxylase (EC 1.14.11.3) were not inhibited by erythrocyte superoxide dismutase or stimulated by superoxide anion. gamma-Butyrobetaine hydroxylase was inhibited by a number of low-molecular-weight compounds, such as tetranitromethane, Nitro Blue Tetrazolium, adrenaline and Tiron, which may act as scavengers of superoxide anion. Involvement of this radical in other oxygenase reactions has been inferred from the findings that they were inhibitory for the respective enzymes. Several of these compounds also inhibited gamma-butyrobetaine hydroxylase. It could be concluded from these experiments, however, that mechanisms other than disposal of superoxide anion might equally well be operative, such as hydrophobic interaction with the enzyme protein and interaction with compounds required for full enzymic activity, e.g. iron and ascorbate. The results appear to rule out a requirement for superoxide anion generated in free solution, and have not yielded evidence for participation of enzyme-bound superoxide anion in 2-oxoglutarate-dependent hydroxylations.
Topics: Copper; Hydroquinones; Hydroxylation; Ketoglutaric Acids; Mixed Function Oxygenases; Nitroblue Tetrazolium; Oxygen; Quinones; Superoxide Dismutase; Superoxides; Tetranitromethane; gamma-Butyrobetaine Dioxygenase
PubMed: 6291507
DOI: 10.1042/bj2050339 -
The Journal of Biological Chemistry Jul 1983We have chemically modified groups of amino acids in the sequence of alpha-cobratoxin and have studied the derivatives as to their affinity of binding to the...
We have chemically modified groups of amino acids in the sequence of alpha-cobratoxin and have studied the derivatives as to their affinity of binding to the acetylcholine receptor protein from Torpedo marmorata. (i) The toxin derivatives which were fully modified at lysine (penta-epsilon-N,N-dimethyl lysine; penta-epsilon-N-acetyl lysine), arginine (penta-N7,N8-(1,2-dihydroxycyclohex-1,2-ylene arginine), and tyrosine (mononitrotyrosine) all had significant remaining toxicity and affinity of binding. (ii) The "extra" disulfide of alpha-cobratoxin was selectively reduced and alkylated. Depending on the charge, size, and hydrophobicity of the attached groups, derivatives were obtained that bound to the acetylcholine receptor with higher (di-S-carboxyamidomethyl), about equal (di-S-pyridylethyl), or lower (di-iodoacetaminoethylnaphthylamine-5-sulfonic acid) affinity than the unmodified toxin. (iii) A fully reduced and carbamidomethylated derivative of alpha-cobratoxin obtained by repeating the procedure for selective reduction six times still bound with appreciable affinity (KD approximately 3 X 10(-6) M) to the acetylcholine receptor. We conclude that neither a single positively charged residue nor tyrosine nor the integrity of the disulfides is absolutely essential for toxicity. Furthermore, the single tyrosine and the area around the extra disulfide do not participate in the binding to the receptor. Together with previous findings on this interaction, this suggests a multipoint attachment of toxin and receptor involving several locally separate structural elements of the toxin.
Topics: Acylation; Amino Acid Sequence; Animals; Arginine; Cobra Neurotoxin Proteins; Cyclohexanones; Elapid Venoms; Kinetics; Methylation; Mice; Pancreatic Elastase; Peptide Fragments; Receptors, Cholinergic; Tetranitromethane; Torpedo; Tyrosine
PubMed: 6553056
DOI: No ID Found -
The Journal of Biological Chemistry Mar 1994The specificity and active site properties of vitamin B6 phosphatase purified from human erythrocytes were studied by kinetic analyses with vitamin B6 compounds and...
The specificity and active site properties of vitamin B6 phosphatase purified from human erythrocytes were studied by kinetic analyses with vitamin B6 compounds and derivatives and chemical modification with group-specific reagents. The kinetic constants for pyridoxal phosphate (PLP), 4-pyridoxic acid phosphate, pyridoxine phosphate, and pyridoxamine phosphate were determined from pH 5 to 9. The values of Vmax/Km and pKm were highest for PLP and 4-pyridoxic acid phosphate and lowest for pyridoxamine phosphate. Vmax/Km and pKm for the four substrates were maximum between pH 6 and 8. Ionizable groups with pKa values about 6 and 8 affected substrate binding to the enzyme. Vmax values for all the substrates gradually decreased with increasing pH. The enzyme also catalyzed the dephosphorylation of 4'-secondary amine derivatives of vitamin B6-phosphate. The phosphatase had greatest catalytic efficiency with substrates that contained a negatively charged group on the 4'-position of the pyridine ring. It is concluded that there are one or two positively charged groups at the active site of the enzyme that interact with the substrate's phosphate ester and 4'-substituent. The phosphatase was inactivated by phenylglyoxal, and PLP protected the enzyme against this inactivation. Phenylglyoxal did not modify Lys or Cys residues or an alpha-amino group since the enzyme's NH2 terminus is blocked, and it did not affect the quaternary structure of the phosphatase. The enzyme was inactivated by the incorporation of 1 mol of phenylglyoxal/subunit. Diethylpyrocarbonate inactivated the enzyme by reacting with a group with a pKa of 6.7, and pyridoxine phosphate protected the enzyme against this inactivation. These data suggest that Arg and His residues are at or near the active site and may play roles in substrate binding and/or catalysis.
Topics: Diethyl Pyrocarbonate; Erythrocytes; Humans; Kinetics; Phenylglyoxal; Phosphoric Monoester Hydrolases; Substrate Specificity; Tetranitromethane
PubMed: 8125926
DOI: No ID Found -
The Journal of Biological Chemistry Mar 1988Previous studies from several laboratories have shown that thrombin is inactivated by tetranitromethane with the formation of nitrotyrosine. The inactivation is...
Previous studies from several laboratories have shown that thrombin is inactivated by tetranitromethane with the formation of nitrotyrosine. The inactivation is characterized by an apparently greater loss of fibrinogen-clotting activity than activity toward synthetic ester substrates, suggesting that the residues modified by tetranitromethane are involved in the interaction of thrombin with fibrinogen. This study was designed 1) to determine the effect of solvent conditions on the rate of modification and the stoichiometry of the reaction of tetranitromethane with bovine alpha-thrombin; 2) to identify the residue(s) modified; and 3) to characterize the modified enzyme with respect to its interaction with peptide nitroanilide substrates and fibrinogen. The inactivation of thrombin by tetranitromethane proceeded more rapidly in 50 mM Tris, pH 8.0, than in 50 mM sodium phosphate, 100 mM NaCl, pH 8.0. Approximately 10% fibrinogen-clotting activity remained at maximal inactivation. A study of the effect of tetranitromethane concentration on the rate of inactivation suggested that the loss of activity was the result of the modification of 1 mol of tyrosine/mol of thrombin. A similar result was obtained from the analysis of the extent of inactivation as a function of the extent of protein modification. Structural analysis of the modified protein showed substantial modification at both Tyr71 and Tyr85. Enzyme kinetic studies were performed with the modified protein and a control thrombin with N2-tosylglycylprolylarginine p-nitroanilide. H-D-phenylalanylpipecolylarginine p-nitronailide, and purified bovine fibrinogen. With all three substrates, a substantial decrease in kcat was observed, whereas there was essentially no change in Km. These results suggest that, contrary to previous suggestions, the modification of Tyr71 and Tyr85 in thrombin does not influence the binding of substrates, but rather influences active site reactivity.
Topics: Animals; Cattle; Kinetics; Methane; Peptide Fragments; Peptide Mapping; Protein Binding; Solvents; Substrate Specificity; Tetranitromethane; Thrombin; Trypsin
PubMed: 3346219
DOI: No ID Found