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PubMed1995

Chemical modification of the urokinase-type plasminogen activator and its receptor using tetranitromethane. Evidence for the involvement of specific tyrosine residues in both molecules during receptor-ligand interaction.

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid anchored receptor (uPAR) is essential for the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metastasis. The receptor binding site of uPA is retained within its isolated growth factor-like module (GFD; residues 4-43). The NH2-terminal domain of uPAR has a primary role in uPA binding, although maintenance of its multidomain structure has been shown to be necessary for the high affinity of this interaction [Ploug, M., Ellis, V., & Danø, K. (1994) Biochemistry 33, 8991-8997]. To identify residues engaged in the uPAR-uPA interaction, we have performed a "protein-protein footprinting" study on preformed uPAR-GFD complexes by chemical modification with tetranitromethane. All six tyrosine residues in uPAR and the single tyrosine residue in GFD (Tyr24) were susceptible to nitration in the native uncomplexed proteins, whereas in the receptor-ligand complexes both Tyr57 of uPAR and Tyr24 of GFD were protected from modification. Modification of uPAR alone led to a parallel reduction in the potential to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsic fluorophore reporting on the accessibility of a hydrophobic site involved in uPA binding. These data clearly demonstrate that Tyr57 in the NH2-terminal domain of uPAR and Tyr24 in uPA are intimately engaged in the receptor-ligand interaction, whereas Tyr87 positioned in the linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface.

Amino Acid Sequence↗

PubMed1985

Increased agonist affinity is induced in tetranitromethane-modified muscarinic receptors.

Tetranitromethane (TNM) modifies the muscarinic receptors from rat cerebral cortex. The modified receptor possesses an increased binding affinity (6-9-fold) toward several agonists such as acetylcholine, carbamoylcholine, arecoline, etc. The binding of antagonists (Bmax and Kd) is only slightly altered. The effects of TNM treatment can be prevented by atropine, thus indicating that TNM modifies residue(s) at the binding site. We carried out a series of successive chemical modifications which indicated that the modified residue(s) is (are) most probably a tyrosyl and not a cysteinyl residue. This conclusion gains support from the pH profile of agonist binding, which suggests the involvement of a residue with an apparent pK comparable to that of the phenolic hydroxyl of a nitrotyrosyl residue. The binding properties of the modified receptor, when compared to those of the native one, clearly indicate that the response to TNM modification with respect to the binding of agonists such as acetylcholine and carbamoylcholine is different from that when oxotremorine and its analogue are employed. This is interpreted as being the result of different binding modes exhibited by the various agonists. Nitration of the receptors can be prevented by the presence of an antagonist but not by an agonist. We propose that this differential response is due to the formation of ligand-receptor complexes that differ with respect to the microenvironment of the modified tyrosyl residue.

Acetylcholine↗

PubMed1975

Histone-histone associations within chromatin. Cross-linking studies using tetranitromethane.

Treatment of chromatin with the protein cross-linker tetranitromethane (TNM) results in a product identified as an F2a1-F2b dimer. The same product appears after treatment with TNM of HeLa cells growing in culture. Furthermore acid-extracted histones which have been fractionated into the five separate species can be recombined and mixed with DNA to produce a nucleohistone preparation which is also cross-linked by TNM to give the F2a1-F2b dimer. F1 and F3 can be excluded from the reconstitution mixture without effect on the dimer production. In contrast, the presence of F2a2 is essential to the proper reconstitution of F2a1 and F2b with DNA. The specificity of TNM and the characteristics of the reaction suggest that F2a1 and F2b are cross-linked at their specific binding sites. These results provide evidence that F2a1, F2a2, and F2b interact specifically in chromatin.

Animals↗

PubMed2006

Tyrosine-dependent oxidative DNA damage induced by carcinogenic tetranitromethane.

Tetranitromethane (TNM) is used as an oxidizer in rocket propellants and explosives and as an additive to increase the cetane number of diesel fuel. TNM was reported to induce pulmonary adenocarcinomas and squamous cell carcinomas in mice and rats. However, the mechanisms underlying carcinogenesis induced by TNM has not yet been clarified. We previously revealed that nitroTyr and nitroTyr-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Since TNM is a reagent for nitration of Tyr in proteins and peptides, we have hypothesized that TNM-treated Tyr and Tyr-containing peptides induce DNA damage by the modification of Tyr. We examined DNA damage induced by TNM-treated amino acids or peptides using (32)P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. TNM-treated Tyr and Lys-Tyr-Lys induced DNA damage including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of Cu(II) and NADH. DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). The cytosine residue of the ACG sequence complementary to codon 273, well-known hotspots of the p53 gene, was cleaved with piperidine and Fpg treatments. On the other hand, nitroTyr and Lys-nitroTyr-Lys did not induce DNA damage in the presence of Cu(II) and NADH. Time-of-flight mass spectrometry confirmed that reactions between Lys-Tyr-Lys and TNM yielded not only Lys-nitroTyr-Lys but also Lys-nitrosoTyr-Lys. Therefore, it is speculated that the nitrosotyrosine residue can induce oxidative DNA damage in the presence of Cu(II) and NADH. It is concluded that Tyr-dependent DNA damage may play an important role in the carcinogenicity of TNM. TNM is a new type of carcinogen that induces DNA damage not by itself but via Tyr modification.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1978

Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking.

1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group.

Amino Acids↗

PubMed1987

Chemical modification of human alpha 1-proteinase inhibitor by tetranitromethane. Structure-function relationship.

Nitration of tyrosine residues of alpha 1-proteinase inhibitor (alpha 1-PI) by tetranitromethane yielded a product that maintained its inhibitory activity against trypsin but lost most of its inhibitory activity against elastase. Chemical analysis of the product showed that four out of the six tyrosine residues in alpha 1-PI had been nitrated to various degrees: Tyr-38 and Tyr-297 were not nitrated, whereas Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were nitrated to extents in the range 40-80%. We interpreted these data to mean that modification of these tyrosine residues decreased the association constant between alpha 1-PI and the proteinases and that the decrease differs from one proteinase to the other. When either alpha 1-PI-trypsin or alpha 1-PI-elastase complex was nitrated, nitration took place only to a very slight extent at these latter four tyrosine residues. On the other hand, Tyr-38 and Tyr-297 underwent nitration to about 20%. We concluded that Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were located on the surface of alpha 1-PI that interacts with either trypsin or elastase in the formation of complexes, and were therefore protected from nitration.

Amino Acids↗

PubMed1982

Yields of hydroxyl radical and reducing species in tritiated water based on the radiolysis of tetranitromethane.

Yields of the nitroform anion produced in tetranitromethane aqueous solutions including tritiated water with and without isopropanol were measured. Yields of .OH and reducing species (e-aq + .OH) were calculated from the yields of the nitroform anion. The G values obtained for them were 3.8 and 2.5, respectively. The former was higher and the latter was lower than those for 60Co gamma-rays. Their beta/gamma ratio was 1.36 and 0.76, respectively. The beta/gamma ratio, 1.36, for .OH may be related to r.b.e. values higher than the one reported for 3H beta-rays.

Cobalt Radioisotopes↗

PubMed1987

Hydroxyl radical-induced strand break formation of poly(U) in anoxic solution. Effect of dithiothreitol and tetranitromethane.

The role of dithiothreitol (DTT) and tetranitromethane (TNM) on the yields of radiation-induced strand break formation in polyuridylic acid (poly(U] was studied in anoxic aqueous solutions at neutral pH by low-angle laser light-scattering. From G (single-strand breaks) as a function of DTT concentration it follows that two different processes lead to OH radical-induced single-strand break (ssb) formation. Only one of the two processes, which accounts for 80 per cent of the ssb formation, is inhibited by DTT, the other one, 20 per cent, is not inhibited. The 'repair' process is attributed to H-donation to the C-6-yl radical of the uracil moiety. The C-6-yl radical is produced by OH addition to the C-5 position of the uracil moiety. It follows that the sugar radicals, in contrast to earlier suggestions, do not seem to be repaired by DTT at the low concentrations used. The strand break formation not inhibited by DTT is induced by radicals other than the uracil-6-yl radical, e.g. the uracil-5-yl or the OH radicals reacting with the sugar moiety. The strong reduction of G(ssb) from 2.3 to 0.2 on addition of TNM is also discussed.

Chemical Phenomena↗

PubMed2000

Superoxide, amine buffers and tetranitromethane: a novel free radical chain reaction.

The amine buffer Tris slowly reduces tetranitromethane (TNM) to the nitroform anion in a non-accelerating reaction. The amine buffers HEPES and MOPS also (slowly) react with TNM but the dialkylaminoalkyl radicals formed from these two buffers undergo further reactions resulting in a rapid, accelerating, free radical chain process whereby the amine is oxidized and TNM reduced. The chemical functionality in any reaction component, not necessarily the buffer, required for this radical chain mechanism is >N-CH<. In the presence of such groups, the quantification of superoxide by TNM is impossible.

Amines↗

PubMed1989

Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated.

A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.

Amino Acid Sequence↗

PubMed1990

Reaction of hen egg-white lysozyme with tetranitromethane: a new side reaction, oxidative bond cleavage at glycine 104, and sequential nitration of three tyrosine residues.

We found that the reaction of hen egg-white lysozyme with an equimolar amount of tetranitromethane (TNM) at pH 8.0 and room temperature yielded derivatives in which the N-C bond of Gly104 is oxidatively cleaved, and a mono-nitrotyrosine lysozyme in which Tyr23 is nitrated. This bond cleavage occurred more predominantly with a decrease in the nitration of Tyr23, when the reaction was carried out under more dilute conditions. A possible mechanism in which a phenoxyl radical of Tyr 23 (an intermediate of nitration) is involved was proposed for this oxidative bond cleavage. When lysozyme was reacted with a 10 times molar excess of TNM, in addition to a mono-nitrotyrosine lysozyme in which only Try23 is nitrated, a di-nitrotyrosine lysozyme in which Tyr20 and Tyr23 are both nitrated and a tri-nitrotyrosine lysozyme in which Tyr20, Tyr23, and Tyr53 are all nitrated were obtained. However, no other possible mono- or di-nitrotyrosine lysozymes could be isolated. Thus, it is concluded that the three tyrosine residues in lysozyme are essentially nitrated sequentially with TNM in the order of Tyr23, Tyr20, and Tyr53. Since the derivatives obtained here were all active, none of the three tyrosine residues or the residues around Gly104 are considered to be very important for the lysozyme activity.

Animals↗

PubMed1978

Modification of papain with tetranitromethane.

Papain [EC 3.4.22.2] polymerizes readily upon treatment with tetranitromethane (TNM) by forming intermolecular covalent linkages through its tyrosine residues (Tsukamoto, S. & Ohno, M. (1974) J. Biochem. 75, 1377-1380). Polymerization occurred optimally at pH 9.0 with S-sulfenylsulfonate papain. Circular dichroic spectra of polymerized papains showed a small change in ellipticity when compared with that of unmodified papain. Esterolytic activity of the modified enzyme toward benzoyl-L-arginine ethyl ester (BAEE) was almost fully retained, at least up to the formation of hexamer, with an unchanged Km value. Spectrophotometric and amino acid analyses indicated that two or three tyrosine residues are involved in intermolecular crosslinks depending on the amount of TNM used. The tyrosine residues nitrated were identified as those at positions 61, 116, 103, and 4, the extent of nitration decreasing in this order. When activated papain was treated with increasing molar ratios of TNM, an essential sulfhydryl function was first oxidized and, at a 2-fold molar excess of the reagent, restoration of activity was no longer observed even after addition of dithiothreitol (DTT). The evidence obtained in the present study eliminates the possibility of inactivation due to nitration of a tryptophan residue, which had been suggested previously.

Amino Acid Sequence↗

PubMed1977

Reaction of bovine and equine growth hormones with tetranitromethane.

Bovine and equine growth hormones were chemically modified with tetranitromethane, at pH 7.4 during 5 h and at pH 8.0 in the presence of 8 M urea during 1 h. a) Both hormones have very similar but not identical reactivities. b) The nitration of the reactive tyrosines and tryptophan residues at pH 7.4 produces no detectable changes in their immunological or somatotrophic activities. C) The nitration of all tyrosine residues in both hormones gives rise to a complete loss of somatographic activity with no alteration of the immunological activity.

Amino Acids↗

PubMed1981

Modification of tryptophanase with tetranitromethane.

Modification of apotryptophanase with tetranitromethane [C(NO2)4] resulted in a loss of enzymatic activity, whereas holotryptophanase was highly resistant against C(NO2)4-inactivation. The essential importance of the active-site-bound pyridoxal 5'-phosphate (pyridoxal-P) for the protection was confirmed by the agreement of K 1/2 (protection) (1.2 microM) for pyridoxal-P with Km (1.5 microM) in enzyme catalysis. Amino acid analyses and inactivation stoichiometry showed that modification of 1--2 tyrosyl residues per monomer caused complete inactivation. The appearance of 430-nm species upon incubation of C(NO2)4-inactivated apoenzyme with pyridoxal-P indicated that the C(NO2)4-inactivated apoenzyme could still bind the coenzyme, although an affinity of the enzyme for pyridoxal-P (Kd = 51 microM) was much lower than that of the native enzyme (Kd = 0.7 microM). A close relationship was observed between the cofactor activity of monovalent cations and their effectiveness in the protection by pyridoxal-P: in the presence of active monovalent cations (K+, NH+4 and Rb+) pyridoxal-P could provide the protection but not in the presence of inactive cations (Li+, Na+ and Cs+) as well as in the absence of inorganic monovalent cations. From the experimental results obtained it was suggested strongly that tryptophanase has essential tyrosyl residues near the active site. The tyrosyl residues were prevented from the attack of C(NO2)4 by the active-site-bound pyridoxal-P only in the catalytically active holoenzyme.

Amino Acids↗

PubMed1984

Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state.

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.

Bacillus megaterium↗

PubMed1987

Chemical modification of phosphorylase b by tetranitromethane. Identification of a functional tyrosyl residue.

Tetranitromethane, C(NO2)4, a reagent for tyrosyl residues, was found to inactivate irreversibly rabbit skeletal muscle glycogen phosphorylase b. Under the chosen conditions seven tyrosyl residues, namely Tyr-75, 203, 262, 280, 403, 552 and 647, were found to be nitrated. Inactivation was prevented by the presence of the allosteric activator 5'-AMP during nitration. Under these latter conditions one of the reactive tyrosyl residues was not modified by C(NO2)4; thus, this residue appeared to be essential for either catalytic activity or allosteric activation. Tryptic digests of phosphorylase b, reacted with C(NO2)4 in the absence and presence of 5'AMP, were fractionated by gel filtration. The peptide mixtures were further purified by reverse-phase HPLC. One of the peptides contained the tyrosyl residue which was modified by C(NO2)4 only in the absence of 5'AMP. The sequence of this peptide was determined. The amino acid residue which is responsible for the loss of activity upon reaction with C(NO2)4 was identified in the amino acid sequence of phosphorylase b as tyrosine-75. Of the other residues modified in the presence and in the absence of C(NO2)4, tyrosine-403 contributes to the glycogen-storage site whereas Tyr-280 is close to the alpha-D-glucose-binding site. These residues, exposed to the solvent both in the presence and in the absence of 5'AMP, are not essential for catalytic activity.

Amino Acid Sequence↗

PubMed1988

Tetranitromethane modification of the muscarinic receptors in bovine adrenal medulla.

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.

Adrenal Medulla↗

PubMed1978

[Reaction between tetranitromethane and deoxyribonucleoproteins].

A comparative kinetic study has been made of tetranitromethane nitration of tyrosine residues in deoxyribonucleoprotein preparation (DNP) treated with EDTA and/or UV light at lambda=253.7 nm, as well as obtained by enzymatic digestion of nucleosomes. UV-light-induced protein-DNA linkages stabilize the structure of the preparation, whereas the action of chelate agents causes DNP-200 angstrom leads to DNP-100 angstrom transfer. Comparison of kinetic data and the results of the amino acid analysis of individual fractions of nitrated histones allowed to conclude that the differences observed between the degree and the rate of nitration are due to internucleosomic interactions which form the supernucleosomic structure of DNP.

Chemical Phenomena↗