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1.
The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of 125I-labeled toxin B-IV by native toxin B-IV have shown specific binding of 125I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [3H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association () and dissociation () shows a nearly identical of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding. 相似文献
2.
A synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV has been designed, cloned, and expressed in Escherichia coli. Although expression of the toxin alone appears to be incompatible with host viability, large amounts could be synthesized as a fusion protein with either E. coli beta-galactosidase or the gene 9 protein of bacteriophage T7, the latter system being the more efficient. The fusion protein has been purified, and, after Factor Xa-catalyzed hydrolysis at a customized linker site, we have obtained the equivalent of 12 mg of pure toxin B-IV per liter of bacterial culture. The recombinant protein is identical with B-IV isolated from Cerebratulus with respect to high performance liquid chromatography mobility and secondary structure, and its amino acid composition differs only by the presence of an amino-terminal methionine residue and replacement of Hyp10 by Pro. Quantal bioassay indicates that the cloned protein is comparable to the natural toxin in specific toxicity. The small differences observed in comparing the activities of the two proteins are most likely due to the presence of the methionine extension at the amino terminus of the recombinant, although lack of hydroxylation of Pro10 may also contribute. 相似文献
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Kenneth M. Blumenthal 《Archives of biochemistry and biophysics》1980,203(2):822-826
Reaction of Cerebratulus lacteus toxin B-IV with 2-hydroxy-5-nitrobenzyl bromide at pH 4.5 results in modification of toxin tryptophan residues and loss of biological activity. With relatively small reagent excesses, one tryptophan per molecule is modified without major effect on toxicity. Further reaction results in modification of a second residue of tryptophan and loss of at least 95% of the toxic activity. Modification of one or both tryptophan residues is without significant effect on the secondary structure of the protein. The specificity of each phase of the reaction has been assessed by fingerprint analysis of peptides derived from toxin modified to differing extents with 2-hydroxy-5-nitrobenzyl bromide. It is thus possible to show that tryptophan-5 reacts first and tryptophan-30 only under more rigorous conditions. It thus appears that tryptophan-30 is essential for full neurotoxic activity. 相似文献
5.
Neurospora glutamate dehydrogenase (NADP-specific) is rapidly inactivated upon reaction with tetranitromethane. This inactivation is completely prevented by the presence of coenzyme (NADP) or nicotinamide mononucleotide (NMN) but not by substrate. NADH, or 2'-monophosphoadenosine-5'-diphosphoribose. Amino acid analysis indicates that the primary effect of modification is nitration of a single residue of tyrosine per polypeptide chain. We have identified the reactive tyrosine by isolation of a single, uniquely labeled peptide after hydrolysis with trypsin followed by cleavage with cyanogen bromide. The modified residue proved to be tyrosine-168 in the linear sequence. This residue is not present in the part of the sequence that had been previously implicated as involved in the binding of the adenylate portion of the coenzyme. Both NMN and 2-monophosphoadenosine-5'-diphosphoribose act as competitive inhibitors of NADP in the oxidation of glutamate with Ki values of 4.65 x 10(-4) M and 4.30 x 10(-4) M, respectively. Thus, the specific protection afforded by NADP and NMN, but not by 2'-monophosphoadenosine-5'-diphosphoribose, indicates that tyrosine-168 is involved in binding the nicotinamide portion of the coenzyme. 相似文献
6.
William R. Kem Chiang-Kuang Tu Robert W. Williams Arazdordi Toumadje W. Curtis Johnson Jr. 《The protein journal》1990,9(4):433-443
The secondary structure ofCerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical α-helical peaks at 191, 208, and 222 nm. At neutralpH, the α-helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% α-helix estimate. Both CD and Raman spectroscopic methods failed to detect any β-sheet structure. The spectroscopic analyses revealed significantly more α-helix and less β-sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49–55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing thatCerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at highpH are consistent with this interpretation. 相似文献
7.
Hao Zhang 《Archives of biochemistry and biophysics》2009,484(2):134-618
Recent reports suggest that intramolecular electron transfer reactions can profoundly affect the site and specificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects of methionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H2O2 and NO2− and peroxynitrite (ONOO−) or ONOO− and bicarbonate (HCO3−) in model peptides, tyrosylmethionine (YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides were analyzed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediates were identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown (Zhang et al., J. Biol. Chem. 280 (2005) 40684-40698) that oxidation and nitration of tyrosyl residue was inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show that methionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighboring tyrosine residue in the presence of ONOO− (or ONOOCO2−) or MPO/H2O2/NO2− system. Nitration of tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO− (formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF, YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteine residues have been attributed to differences in the direction of intramolecular electron transfer in these peptides. Further support for the interpretation was obtained by steady-state radiolysis and photolysis experiments. Potential implications of the intramolecular electron transfer mechanism in mediating selective nitration of protein tyrosyl groups are discussed. 相似文献
8.
Tetanus toxin acts by blocking the release of glycine from inhibitory neurones within the spinal cord. An initial stage in the toxin's action is binding to acceptors on the nerve surface and polysialogangliosides are a component of these acceptor moieties. Using site-directed mutagenesis, we identify tyrosine-1290 of tetanus toxin as a key residue that is involved in ganglioside binding. This residue, which is located at the centre of a shallow pocket on the beta-trefoil domain of the tetanus H(c) fragment, is also shown to play a key role in the functional binding of tetanus toxin to spinal cord neurones leading to the inhibition of neurotransmitter release. 相似文献
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Victor S. Sharov Elena S. Dremina Christian Schöneich 《Archives of biochemistry and biophysics》2009,484(2):155-762
There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function [Sharov et al., Exp. Gerontol. 41 (2006) 407-416]; in another report, the nitration of one specific residue, Tyr613, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation [Dairou et al., J. Mol. Biol. 372 (2007) 1009-1021]. In this study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite. 相似文献
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《Biochimica et Biophysica Acta.Protein Structure》1976,420(2):265-278
Of the three tyrosine residues available for nitration by tetranitromethane in hemerythrin, nitration of tyrosine residue 70 has no effect on dissociation of octomers to monomers, but nitration of tyrosines 18 and/or 67 results in dissociation to monomers. The latter data suggests these residues are important for subunit association. The reactive sulfhydryl, the modification of which produces dissociation, was protected as a mixed disulfide during the nitration but was regenerated for analysis of the state of association. Residue 70 can be selectively modified because of its exposed position and perhaps because of its slightly lower pKa of 6.9, compared to 7.3 as an average of all nitrotyrosines in a completely nitrated hemerythrin.Solvent perturbation studies in 20% Me2SO indicate that 3 tyrosines, in agreement with the nitration results, and 2 tryptophan residues are exposed; however, oxidation at a 2-fold molar excess of N-bromosuccinimide oxidizes three tryptophan whereas a 3.5-fold excess oxidizes all four, but results in a rapid active site destruction. Photo-oxidation with methylene blue results in oxidation of only two tryptophan residues. These data have been interpreted to indicate that two tryptophans are free and two are involved in subunit association.Photo-oxidation with methylene blue results in the destruction of three histidines but no decrease in active site absorption. Histidine modification with diethyloxydiformate shows that three histidines react with no change in active site absorption. These results indicate that four histidines are unreactive toward these modifying agents and are therefore either buried or are ligands to the iron. 相似文献
13.
In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H+ conduction and other reactions catalyzed by the H+-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-33Pi exchange, and proton conduction by the H+-ATPase complex. These effects are caused by nitration of tyrosine residues of H+-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-33Pi exchange in the complex purified from tetranitromethane-treated particles. H+ conduction in phospholipid vesicles inlaid with F0 is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F0 are critically involved in energy-linked proton translocation in the ATP-ase complex. 相似文献
14.
Pavlov E Britvina T McArthur JR Ma Q Sierralta I Zamponi GW French RJ 《Biophysical journal》2008,95(9):4277-4288
External μ-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two μ-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the μ-conotoxin and the DEA-binding site of ∼15 Å. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by ∼4-fold; and 2), increasing external [Na+] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions. 相似文献
15.
Gary Strobel Neal Van Alfen Kenneth D. Hapner Michael McNeil Peter Albersheim 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,538(1):60-75
Ceratocystis ulmi, the causal agent of Dutch Elm Disease, produces phytotoxic glycopeptides in culture. A mixture of phytotoxic glycopeptides has been prepared by affinity chromatography on a concanavalin A-Sepharose column and collectively they have been termed the toxin. The polydisperse component that makes up the majority of the toxin (80%) by weight has a molecular weight of about 2.7·105. The large molecular weight component (<5%) elutes at the void volume of a Bio-Gel A50 m column. The other component (15%) appears as a trailing peak on the edge of the major component and has an approximate molecular weight of 7 · 104. The toxin is composed of 38% sugar residues, primarily rhamnose and mannose, and 7% amino acid residues. Methylation analysis coupled with mild acid hydrolysis indicates that the backbone of the polysaccharide portion of the toxin is composed of α-1,6-linked mannosyl residues with a 3-linked terminal rhamnosyl residue linked to C-3 of almost every mannosyl residue. The carbohydrate portion of the molecule is linked to the peptide via O-glycosidic linkages to both threonyl and seryl residues. All three components of the toxin are capable of causing wilt in stem cuttings of American elm. 相似文献
16.
John Y. Ng Joyce ChiuPhilip J. Hogg Jason W.H. Wong 《Biochemical and biophysical research communications》2013
Methionyl aminopeptidase 2 (MetAP2) plays an important role in the regulation of angiogenesis. This study examined whether nitration of MetAP2 alters its enzymatic activity in vitro. The activity of unmodified, nitrated and oxidised MetAP2 was assessed and it was found that nitration significantly reduced its ability to cleave a chromogenic substrate. Mass spectrometry analysis identified Tyr336 as a nitrated residue in MetAP2. Structural and evolutionary analysis indicate that this is an important residue for MetAP2 activity. Combined, the results show that the activity of MetAP2 is reduced by nitration and raise the possibility that nitration of MetAP2 is a mechanism contributing to endothelial dysfunction. 相似文献
17.
α-Bungarotoxin (α-Bgt), an α-neurotoxin, has been 14C-methylated by treatment with [14C]formaldehyde following NaCNBH3 reduction. The methylation rate is fast (about 84% methylation in 15 min), with 12 methyl groups incorporated per mole of α-Bgt or a mean of 1.7 methyl groups per available amine residue. The specific activity of α-[14C]Bgt is 768 mCi/mmol. Unlike most of the reported chemical modifications of α-neurotoxins, involving a high decrease of the toxin activity after modification, α-[14C]Bgt retains 100% of its unmodified ability to bind to both isolated acetylcholine receptor (AcChR) and AcChR-enriched membrane fragments prepared from Torpedo californica. This lysyl residue modification does not perturb the toxin binding activity, probably, because the net positive charges of the ?-amino groups and amino-terminal residue remain unaltered. 14C-Methylated α-Bgt appears better suited than 125I-α-Bgt for use in AcChR binding studies because of the longer half-life of the isotope, and the apparent high uniformity of labeling of the toxin preparations. 相似文献
18.
Peter Scherrer Lester Packer Stanley Seltzer 《Archives of biochemistry and biophysics》1981,212(2):589-601
Bacteriorhodopsin in the purple membrane of Halobacterium halobium is coupled to a photocycle that results in the release and uptake of protons. The role of tyrosyl residues in the photocycle of bacteriorhodopsin has been investigated by the technique of chemical modifications of these residues by iodination and nitration. The studies indicate that modification of a tyrosyl residue accelerates M412 formation, whereas modification of another type of tyrosine residue(s) accessible from the cytoplasmic surface of the purple membrane inhibits M412 decay. The results support the hypothesis that a reversible deprotonation of tyrosine residues prior to and after M412 formation in the photocycle are steps in the light-driven pathway of H+ translocation by bacteriorhodopsin. 相似文献
19.
The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca2+ gradients, induced by the toxin in T corn was prevented by N,N′-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N′-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site. 相似文献
20.
W.E. Thomas James G. Townsel 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,632(4):598-610
The binding of 125I-labeled α-bungarotoxin in the central nervous system of the horseshoe crab, Limulus polyphemus, was investigated. Comparative binding studies in various tissues of L. polyphemus demonstrated a selective association of the toxin with nervous tissues. The greatest enrichment of toxin binding in subcellular fractions of brain tissue was observed in a fraction enriched in mitochondria and acetylcholinesterase-containing membranes. Autoradiographic studies revealed the localization of α-bungarotoxin binding to the longitudinal connectives and neuropile regions of the abdominal ganglia. Three toxin binding components with approximate sedimentation coefficients of 9 S, 15.4 S and 17.4 S were present in solubilized extracts of brain tissue. 125I-labeled α-bungarotoxin binding to these components was inhibited 72%, 47%, 9% and 0% by 10 μM concentrations of (+)-tubocurarine, nicotine, scopolamine and pilocarpine, respectively. The apparent formation of the 15.4 S and 17.4 S proteins from the 9 S protein was obtained. The 15.4 S and 17.4 S components are suggested as aggregates of the 9 S protein. This 9 S protein is proposed as an acetylcholine receptor from the central nervous system of L. polyphemus. 相似文献