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Journal of Biochemistry Apr 1997A novel alpha-neurotoxin, Oh-5, was isolated from king cobra (Ophiophagus hannah) venom and purified by successive SP-Sephadex C-25 column chromatography and...
A novel alpha-neurotoxin, Oh-5, was isolated from king cobra (Ophiophagus hannah) venom and purified by successive SP-Sephadex C-25 column chromatography and reversed-phase HPLC. The complete sequence of Oh-5 was determined by Edman degradation of peptide fragments generated by endopeptidases, i.e., trypsin, Saccharomyces aureus V8 protease and lysyl endopeptidase. This novel toxin comprises 72 amino acid residues with 10 cysteines. The sequence shows 89% sequence homology with Oh-4, and 60% with Toxins a and b from the same venom. The tyrosine, tryptophan, lysine and arginine residues in Oh-5 were modified with tetranitromethane (TNM), 2-nitrophenylsulfenyl (NPS) chloride, trinitrobenzene sulfonate (TNBS), and p-hydroxyphenylglyoxal (HPG), respectively. Modification of Tyr-4 or Trp-27 did not affect the lethal toxicity at all, while the Tyr-4 and 23 nitrated derivative retained about 50% of the lethality of native toxin. Selective trinitrophenylation of Lys-51 or 69 resulted in a decrease in lethality by 29%, and 50% lethality was retained after modification of Lys-2, 51, and 69. A drastic decrease in lethality to 26% was observed when both Arg-35 and 37 were modified. The neurotoxicity was further decreased when Arg-9 was additionally modified. These results suggest that the aromatic residues, Tyr-4 and Trp-27, are not crucial for the neurotoxicity, whereas the cationic residues are involved in multipoint contact between the toxin molecule and the nicotinic acetylcholine receptor (nAChR). The residues Tyr-23 and Arg-35 and 37 in the central loop of Oh-5 seem to contribute greatly to the neurotoxicity.
Topics: Amino Acid Sequence; Animals; Arginine; Chromatography, High Pressure Liquid; Circular Dichroism; Cobra Neurotoxin Proteins; Lethal Dose 50; Mice; Molecular Sequence Data; Sequence Analysis; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Structure-Activity Relationship
PubMed: 9163519
DOI: 10.1093/oxfordjournals.jbchem.a021641 -
FEBS Letters Mar 1986Tetranitromethane reacts with the uncoupling protein of intact brown fat mitochondria. The chloride permeability in the absence of the inhibitory nucleotide GDP is not...
Tetranitromethane reacts with the uncoupling protein of intact brown fat mitochondria. The chloride permeability in the absence of the inhibitory nucleotide GDP is not affected, but the affinity with which GDP binds is decreased, and the coupling between binding of nucleotide and inhibition of chloride permeation is broken.
Topics: Adipose Tissue, Brown; Animals; Binding Sites; Carrier Proteins; Cell Membrane Permeability; Chlorides; Cricetinae; Guanosine Diphosphate; In Vitro Techniques; Ion Channels; Membrane Proteins; Mesocricetus; Methane; Mitochondrial Proteins; Mitochondrial Swelling; Tetranitromethane; Uncoupling Protein 1; Valinomycin
PubMed: 3956727
DOI: 10.1016/0014-5793(86)81178-4 -
The Biochemical Journal Aug 1990The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration,...
The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.
Topics: Chromatography, Gel; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Fibrinolysin; Glutaral; In Vitro Techniques; Macromolecular Substances; Molecular Weight; Peptide Fragments; Poly(ADP-ribose) Polymerases; Protein Denaturation; Structure-Activity Relationship
PubMed: 2144419
DOI: 10.1042/bj2700017 -
Infection and Immunity Apr 1980Cell walls from Streptococcus mutans were prepared by conventional technique and subjected to a series of extraction procedures involving classical protein solvents. The...
Cell walls from Streptococcus mutans were prepared by conventional technique and subjected to a series of extraction procedures involving classical protein solvents. The extracted walls contained several non-peptidoglycan amino acids and were also amenable to radiolabeling with [125I]sodium iodide and chloramine T. The cell walls could be chemically modified with tetranitromethane and diazo-1H-tetrazole, suggesting the presence of tyrosine or histidine or both. Flourescence spectra of the walls revealed the presence of either tyrosine or tryptophan. Several proteases, including pronase, trypsin, subtilisin, and proteinase K, removed some of the label from the walls. In contrast, treatment of the walls with salts or denaturants did not result in the solubilization of label. When the walls were solubilized with mutanolysin and subjected to chromatography, three peaks of radioactivity with apparent molecular weights of 73,000, 39,000, and 9,600 were observed. Wall digests subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band of radioactivity corresponding to an apparent molecular weight of 79,000. Isoelectric focusing of labeled wall digest gave rise to two major bands of radioactivity with isoelectric points of approximately 2.4 and 5.6. The results suggest that the cell wall of S. mutans contains tightly and possibley covalently bound polypeptide molecules. We propose that the cell wall polypeptides of S. mutans serve as factors in the attachment of the bacteria to smooth surfaces.
Topics: Amino Acids; Bacterial Proteins; Cell Wall; Chemical Phenomena; Chemistry; Peptides; Peptidoglycan; Spectrometry, Fluorescence; Streptococcus mutans
PubMed: 7380560
DOI: 10.1128/iai.28.1.118-126.1980 -
International Journal of Molecular... Nov 2023An efficient regioselective approach to novel functionalized bis(isoxazoles) with a variety of aromatic and aliphatic linkers was elaborated, based on the...
An efficient regioselective approach to novel functionalized bis(isoxazoles) with a variety of aromatic and aliphatic linkers was elaborated, based on the heterocyclization reaction of electrophilic alkenes under the treatment with tetranitromethane-triethylamine complex affording 3-EWG-5-nitroisoxazoles. The subsequent reactions of 5-nitroisoxazoles with various ,-, ,- and ,-bis(nucleophiles) provide a wide range of bis(isoxazole) derivatives in good isolated yields. Employing an elaborated method, a series of novel bis(3-EWG-isoxazoles) as the promising allosteric modulators of AMPA receptors were designed and synthesized. The effect of the compounds on the kainate-induced currents was studied in the patch clamp experiments, revealing modulator properties for several of them. The best positive modulator potency was found for dimethyl 5,5'-(ethane-1,2-diylbis(sulfanediyl))bis(isoxazole-3-carboxylate), which potentiated the kainate-induced currents in a wide concentration range (10-10 M) with maximum potentiation of 77% at 10 M. The results were rationalized using molecular docking and molecular dynamics simulations of modulator complexes with the dimeric ligand-binding domain of the GluA2 AMPA receptor. The predicted physicochemical, ADMET, and PAINS properties confirmed that the AMPA receptor modulators based on the bis(isoxazole) scaffold may serve as potential lead compounds for the development of neuroprotective drugs.
Topics: Receptors, AMPA; Kainic Acid; Isoxazoles; Ligands; Molecular Docking Simulation
PubMed: 38003327
DOI: 10.3390/ijms242216135 -
FEBS Letters Mar 1996Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The...
Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.
Topics: Acetic Anhydrides; Acetylation; Amino Acid Sequence; Aspergillus; Enzyme Activation; Enzyme Reactivators; Hydrogen-Ion Concentration; Hydroxylamine; Hydroxylamines; Imidazoles; Molecular Sequence Data; Peptide Fragments; Polygalacturonase; Sequence Analysis; Tetranitromethane; Tyrosine
PubMed: 8612742
DOI: 10.1016/0014-5793(96)00146-9 -
Biochimica Et Biophysica Acta Jun 2004We previously described the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of hemodialyzed patients (HD). The... (Comparative Study)
Comparative Study
We previously described the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of hemodialyzed patients (HD). The present study was carried out to further investigate how myeloperoxidase (MPO)-catalyzed reactions could contribute to AOPP generation in the plasma. First, patterns of plasma protein oxidation obtained after in vitro incubation of control plasma with hypochlorous acid (HOCl) were compared to those from HD patients and control plasma. The use of various analytical techniques enabled localising and identifying the main oxidized proteins with albumin (HSA) after protein separation by size-exclusion chromatography and SDS-PAGE electrophoresis. The characterization of the oxidation level of the individual plasma proteins in terms of carbonyl groups and 3-nitrotyrosine formations was performed by immunoblotting. Secondly, to highlight the significance of AOPP index monitored by spectrophotometry, spectra were established for plasma fractions from HD patients and compared to data for control plasma and HOCl-treated plasma. The corresponding absorbance difference spectra were matched with external standards such as dityrosine, nitrotyrosine and pentosidine and elaborated chromophoric probe models. Indeed, HSA was chlorinated by HOCl reagent or HOCl generated via the MPO/H(2)O(2)/Cl(-) system and was nitrated by tetranitromethane. Increased absorbances at the range of 340 nm were observed both with chlorinated and nitrated HSA. Finally, our results indicate that HOCl, and not NO(2)(*), generated via MPO activity, could represent one of the pathways for AOPP production in plasma proteins exposed to activated phagocytes.
Topics: Biomarkers; Blood Proteins; Humans; Hypochlorous Acid; Nitrites; Oxidation-Reduction; Oxidative Stress; Peroxidase; Renal Dialysis; Renal Insufficiency; Serum Albumin; Spectrophotometry
PubMed: 15196590
DOI: 10.1016/j.bbadis.2004.02.008 -
Japanese Journal of Pharmacology Sep 1988Muscarinic receptor binding was examined in bovine adrenal medullary microsomes following exposure to tetranitromethane (TNM) that modifies tyrosine and cysteine...
Muscarinic receptor binding was examined in bovine adrenal medullary microsomes following exposure to tetranitromethane (TNM) that modifies tyrosine and cysteine residues in proteins. The TNM (10-100 microM) treatment of adrenal medullary microsomes caused a concentration-dependent and irreversible reduction in the maximum number of binding sites (Bmax) for l-(3H)quinuclidinyl benzilate (QNB), with a slight increase in the equilibrium dissociation constant (KD). Typically, about a 36% decrease and a 1.3-fold increase in the corresponding values were obtained at 50 microM of TNM. The alteration in the Bmax was partially prevented by atropine but not carbamylcholine, and it was not reversed by subsequent treatment with dithiothreitol, a disulfide reducing agent. The change in the KD was unaffected by these agents. The TNM (50 microM) treatment also caused a slight decrease in the affinity of atropine and pirenzepine (for both the high and low affinity sites), and it caused a slight decrease in the affinity of carbamylcholine at the high affinity site, with a large loss of the low affinity site. Thus, the results indicate that TNM causes a loss of muscarinic binding sites and a decrease in the binding affinity of muscarinic receptors in bovine adrenal medulla, probably through modifications of functional groups such as tyrosine residues.
Topics: Adrenal Medulla; Animals; Atropine; Carbachol; Cattle; Dithiothreitol; In Vitro Techniques; Kinetics; Methane; Microsomes; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Tetranitromethane
PubMed: 3199607
DOI: 10.1254/jjp.48.67 -
The Biochemical Journal Nov 19811. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the...
1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN'N''-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with alpha-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem.242, 2447-2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.
Topics: Amino Acids; Aminobutyrates; Binding Sites; Chemical Phenomena; Chemistry; Ethyldimethylaminopropyl Carbodiimide; Hemagglutination; Immune Sera; Lectins; Plant Proteins, Dietary; Trisaccharides
PubMed: 7340810
DOI: 10.1042/bj1990399 -
Journal of Biochemistry May 1975In order to study the state of tyrosyl residues in a ribouuclease from bovine semina vesicles [EC 3.1.4.22, RNase Vs1] several lines of experiments were carried out....
In order to study the state of tyrosyl residues in a ribouuclease from bovine semina vesicles [EC 3.1.4.22, RNase Vs1] several lines of experiments were carried out. Spectrophotometric titration of RNase Vs1 indicated that two out of 8 tyrosine residues were titrated very easily and their apparent pKa values were about 9.8. Next, about 4 residues were titrated at pH up to 13.5. The remaining 2 residues were titrated time-dependently at pH 13.5. In 8 M urea, about 6 tyrosine residues were titrated with apparent pK4 values of about 11.2 and about 2 residues were titrated time-dependently at pH 13.5. Acetylation of RNase Vs1 with N-acetylimidazole was studied at pH 7.5. In aqueous solution, about 1.1-3.5 tyrosine residues were acetylated, depending on the experimental conditions, and in 8 M urea, 5.3 tyrosine residues were modified. RNase Vs1 was nitrated with tetranitromethane at pH 7.5. In aqueous solution, about 2.5 tyrosine residues were nitrated very easily; the enzymatic activity of the modified enzymes was 130-200% of that of the native enzyme. In 8 M urea, the reactivity of the tyrosine residues increased and about 4-5.5 residues were modified. The results of chemical modification and spectrophotometric titration indicated that about two tyrosine residues in RNase Vs1 were exposed to the solvent and were more reactive to various reagents, and 3-4 tyrosine residues were less reactive. The final 2 residues were not accessible to the reagent even in the presence of urea, but were titraten at pH 13.5. The solvent perturbation difference spectrum using ethylene glycol as a perturbant indicated that about 4 tyrosine residues were perturbed. When the pH of the enzyme solution was changed from 7.0 to 1.0, the change in optical density of RNase Vs1 due to denaturation blue shift was about 1,600 at 287nm. The optical density change at 287 nm of native RNase Vs1 on exposure to 8 M urea and 6 M guanidine-HCl indicated that the environments of 2-3 and 4 tyrosine residues were changed by the addition of the denaturants, urea and guanidine-HCl, respectively. In RNase Vs1 having about four nitrotyrosine residues, the two most inaccessible tyrosine residues remained resistant to titration with alkali. On adding nucleotide, nitrated RNase Vs1 gave a difference spectrum in the ultraviolet region but not in 320-460 nm region, where nitrotyrosine residues absorb light. This may indicate that tyrosine residues located relatively near the surface of the molecule are not perturbed directly by nucleotide binding.
Topics: Acetylation; Animals; Cattle; Ethylene Glycols; Guanidines; Hydrogen-Ion Concentration; Male; Nucleotides, Cyclic; Protein Conformation; Ribonucleases; Seminal Vesicles; Spectrophotometry; Tetranitromethane; Time Factors; Tyrosine; Uracil Nucleotides; Urea
PubMed: 239931
DOI: 10.1093/oxfordjournals.jbchem.a130808