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PubMed1987

The inactivation of Clostridium perfringens epsilon toxin by treatment with tetranitromethane and N-acetylimidazole.

When one residue of tyrosine per molecule of epsilon toxin was nitrated by tetranitromethane, the modified toxin lost more than 90% of its original activity. The toxin also was inactivated by treatment with N-acetylimidazole. The lethality was restored when the inactive N-acetylimidazole-treated toxin was treated with hydroxylamine. The inactive N-acetylimidazole-treated toxin was not nitrated by tetranitromethane under conditions where one residue of Tyr per molecule of the untreated toxin is nitrated. These data suggest that only one Tyr residue in the toxin is important for the lethal activity.

Acetylation↗
PubMed1979

Amino acid contacts between histones are the same for plants and mammals. Binding-site studies using ultraviolet light and tetranitromethane.

Leek chromatin has been cross-linked by UV light and tetranitromethane. The same major H2A--H2B and H2B--H4 cross-linked dimers are formed as in mammalian chromatin. CNBr peptide mapping shows that the cross-links occur in the same regions of the histone sequence for both plants and mammals. Interspecies complexes formed between leek and calf H2A and H2B can be cross-linked by UV light with the same specificity as intraspecies H2A--H2B complexes. We conclude that certain geometric features of histone-histone binding sites are conserved precisely during evolution despite large changes in the overall histone sequence. Moreover, our data show that identification of cross-linked amino acids using binding-site probes such as UV light and tetranitromethane can yield significant information about thermodynamically important contacts within histone-histone binding sites.

Amino Acids↗
PubMed1979

Effects of modification of the tyrosine residues of bacteriorhodopsin with tetranitromethane.

Treatment of the purple membrane of Halobacterium halobium with tetranitromethane led to modification of tyrosine residues. Modification of more than 3-4 tyrosine residues per bacteriorhodopsin monomer caused a decrease in the light-induced proton-pumping ability of purple membrane in synthetic lipid vesicles, loss of the sharp X-ray-diffraction patterns characteristic of the crystal lattice, loss of the absorbance maximum at 560 nm, and change in the buoyant density of the membrane. No modification of lipid was detected. These changes were interpreted as a gradual denaturation of the protein component such that when 8-9 tyrosine residues are modified, no proton pumping is observed. Modification of less than 3-4 tyrosine residues with tetranitromethane caused an increse in light-induced proton pumping. It was possible to generate partly modified purple membrane which had completely lost the property of diffracting X-rays into the sharp pattern observed with native purple membrane, but which still retained the ability to pump protons in a vectorial manner. Retention of crystal lattice is not essential for proton pumping.

Bacteriorhodopsins↗
PubMed1981

Inactivation of rat muscle 5'-adenylate aminohydrolase by tyrosine nitration with tetranitromethane.

Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues. By amino acid analysis, approximately there tyrosine residues per subunit are estimated to be nitrated in the completely inactivated enzyme. The kinetic properties of the partially inactivated AMP deaminase reveal a negative co-operatively behaviour at approximately half saturation. This suggests that modification of tyrosine residues is also responsible for alteration of the binding properties of the hypothesized activating site of AMP deaminase.

AMP Deaminase↗
PubMed1987

Activation of the K-ras protooncogene in lung tumors from rats and mice chronically exposed to tetranitromethane.

Dominant transforming genes were detected in lung tumors from Fischer 344 rats and C57BL/6 X C3H F1 mice chronically exposed by inhalation to tetranitromethane, a highly volatile compound used in several industrial processes. The rat lung neoplasms were classified as adenocarcinomas, squamous cell carcinomas (epidermoid carcinomas), or adenosquamous carcinomas. The mouse lung tumors were classified as papillary adenocarcinomas or adenomas. In both species, the tumors were morphologically similar to lung tumors in humans. The transfection assay using NIH/3T3 mouse fibroblasts detected transforming genes in 74% (14 of 19) of the rat lung tumors and in 100% (4 of 4) of the mouse lung tumors. Southern blot analysis indicated that transforming gene was an activated K-ras protooncogene in both species. The first exon of the K-ras gene in normal DNA and in DNA from two cell lines transformed by tumor DNA was compared by cloning and sequencing the gene. Experiments showed that there was a GC----AT transition in the second base of the 12th codon of the K-ras oncogene in the two transfectant DNAs. Oligonucleotide hybridization indicated that all of the rat and mouse transfectants had this activating lesion. Additional tumor DNA was then tested for the presence of a mutated allele with the GC----AT transition. All of the rat tumors tested and all of the mouse tumors tested had this mutation present. Hybridization using the normal oligonucleotide sequence around the 12th codon indicated that the normal allele was also present in the majority of the tumors, suggesting that the loss of normal allele is not necessary for the development of neoplasia. One rat lung tumor had no normal allele present, possibly suggesting that this tumor could have been in a more advanced stage than the other tumors. This is the first study to detect activated protooncogenes in rodent tumors induced under conditions which mimic human exposure to a chemical in the workplace. Tetranitromethane may exert its carcinogenic action by both activation of the K-ras oncogene and stimulation of cell proliferation by its irritant properties.

Animals↗
PubMed1987

[Localization of tetranitromethane-modified tyrosine residues in the polypeptide chain of cholesterol-hydroxylating cytochrome P-450].

As a continuation of earlier structure-function relationship studies on cholesterol-hydroxylating cytochrome P-450 from the adrenal cortex mitochondria, the present study deals with the distribution of tetranitromethane-modified tyrosine residues in the hemeprotein polypeptide chain. Amino acid residues Tyr-24, -46, -50, -93, -94, -199, -246 are shown to be modified with tetranitromethane. Tyr-93, -94 are supposedly involved in the active site formation of cytochrome P-450.

Adrenal Cortex↗
PubMed1984

Tetranitromethane oxidation of phytochrome chromophore as a function of spectral form and molecular weight.

Tetranitromethane bleaches Avena phytochrome. The phytochrome (far-red absorbing form; Pfr) chromophore of 124 kilodalton (kD) phytochrome is oxidized 8 times more rapidly than the red absorbing form (Pr). Proteolysis of the 124 kD molecule to the extensively studied mixture of 118 and 114 kD polypeptides increases the rate of oxidation of Pfr 5-fold without affecting the rate of Pr oxidation. As a result, the Pfr form of 118/114 kD preparations is oxidized at a rate 40 times greater than the Pr form. Further proteolytic degradation of the chromoprotein to 60 kD results in an additional increase in the oxidation rates of both Pr and Pfr. These differences in reactivity to tetranitromethane indicate that the chromophore of Pfr is either intrinsically more chemically reactive and/or physically more accessible than the Pr chromophore and that the reactivity/accessibility of both spectral forms is increased by proteolysis. The enhanced reactivity of the Pfr chromophore after proteolytic cleavage of the 6 to 10 kD polypeptide segment(s) from the 124 kD species is further evidence that these segment(s) affect the environment of the native photoreceptor.

Journal Article↗
PubMed1998

Nitration of veratryl alcohol by lignin peroxidase and tetranitromethane.

Lignin peroxidase (LiP), from Phanerochaete chrysosporium, in the presence of H2O2 and tetranitromethane (TNM), oxidizes veratryl (3,4-dimethoxybenzyl) alcohol (VA) (I) to veratraldehyde (IV), 4,5-dimethoxy-2-nitrobenzyl alcohol (V), and 3,4-dimethoxy-nitrobenzene (VI). The formation of these products is explained by a mechanism involving the one-electron oxidation of VA by LiP to produce the corresponding cation radical, which loses a proton to generate the benzylic radical. The latter reduces TNM to generate the trinitromethane anion (VIII) and the nitrogen dioxide radical (.NO2). .NO2 couples with the VA cation radical, and the subsequent loss of a proton leads to V. Alternatively, the attack of .NO2 at C-1 of the VA cation radical, followed by aromatization and loss of formaldehyde (VII), yields VI. Isotopic labeling experiments confirm that V is generated by the reaction of .NO2 with the VA cation radical, rather than with the benzylic radical. The nitration of two other LiP substrates, 1,4-dimethoxybenzene (II) and tyrosine (III), also was examined. Product analysis of reactions conducted in the presence of H2O2 with these substrates indicated less nitrated product was formed from 1,4-dimethoxybenzene and no nitrated product was formed from tyrosine. However, significant amounts of nitrated products were formed from 1,4-dimethoxybenzene and tyrosine when glucose and glucose oxidase were used as an H2O2 source. These results suggest that a reductant, either the veratryl alcohol benzylic radical or superoxide, is required in the reaction to reduce TNM to generate .NO2. These results provide further evidence for the formation of the VA cation radical and the first chemical evidence for the formation of the VA benzylic radical in LiP-catalyzed reactions.

Anisoles↗
PubMed1986

Formation of enzyme-bound carbanion intermediate in the isocitrate lyase-catalyzed reaction: enzymatic reaction of tetranitromethane with substrates and its dependence on effector, pH, and metal ions.

Isocitrate lyase of germinating castor seed endosperm catalyzes the reactions of succinate and of isocitrate (but not of glyoxylate) with tetranitromethane (TNM), giving rise to the nitroform anion (C-(NO2)3), analogous to the reaction of TNM with carbanions (O.P. Malhotra and U.N. Dwivedi, 1984, Ind. J. Biochem. Biophys. 21, 65-67). The kinetics of this reaction have been investigated under a variety of conditions. At a fixed TNM concentration, the initial rate of reaction exhibits a hyperbolic saturation of the enzyme with isocitrate. The reaction with succinate, however, shows "negative cooperativity" in succinate saturation and the data are consistent with the existence of two sets of succinate binding sites of unequal affinity ("tight" and "loose" sites). Equal reaction rates are observed at enzyme-saturating concentrations of succinate and isocitrate. In every case, the rate of reaction is proportional to the TNM concentration. In the presence of alpha-ketoglutarate, hyperbolic saturation curves are obtained for all the substrates (TNM and succinate or TNM and isocitrate). In the presence of this effector the Km of succinate and TNM are independent of the concentration of the second substrate. On the other hand, sets of parallel straight lines are obtained in the double-reciprocal plots for the enzymatic reaction of TNM with isocitrate in the presence of alpha-ketoglutarate. Studies on the effect of pH on the isocitrate lyase-catalyzed reactions of TNM with succinate, TNM with isocitrate, and succinate with glyoxylate in the absence as well as in the presence of alpha-ketoglutarate show that the proton behaves as an uncompetitive inhibitor in all these reactions, suggesting the presence of a "masked" basic group at the enzyme site, which is protonated in the presence of substrate only. The pKa value of this group lies in the range 6.7-6.9. The enzymatic reactions of TNM with succinate and isocitrate exhibit identical Mg2+ ion dependence. From a comparison of the data on the enzymatic reactions of TNM with the corresponding results on the physiological reaction catalyzed by this enzyme, it has been suggested that an ion pair intermediate (E+ X S-, in which E, S, and S- stand for enzyme, succinate, and succinate carbanion, respectively) lies on the pathway of catalysis by isocitrate lyase.

Ricinus communis↗
PubMed1985

Rat brain and heart muscarinic receptors: modification with tetranitromethane.

Tetranitromethane at a concentration of 50 microM modifies the muscarinic receptors in membrane preparations from rat striatum, hippocampus and heart atrium, but not from the rat brain stem. While the binding of antagonists is only slightly altered, the modified receptor possesses an increased affinity of up to 8-fold for [3H]-acetylcholine binding to the high affinity state. This effect is absent if the nitration is carried out in the presence of an antagonist, but not in the presence of an agonist. The affinity for carbamylcholine is increased for both the high and the low affinity state of the receptor, as is evident from its ability to compete with a labeled antagonist. In addition, the proportion of binding sites (alpha) exhibiting the high affinity state for [3H]-acetylcholine or for carbamylcholine is increased upon nitration. This increase cannot be protected against by an antagonist, and is enhanced when nitration takes place in the presence of an agonist. With the agonists oxotremorine and [3H]-oxotremorine-M only the latter effect (i.e., increase in alpha) is observed following nitration, while their dissociation constants for the receptor are unchanged. Data are discussed with respect to the proposed existence of subtypes of muscarinic receptors, as well as the importance of the agonist chosen for studies of ligand-receptor interactions.

Acetylcholine↗
PubMed1986

Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane.

NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.

Animals↗
PubMed1990

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine.

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.

Animals↗
PubMed1982

The reaction of porcine colipase a with tetranitromethane. Generation of extrinsic cotton effects in the visible region.

Porcine pancreatic colipase was reacted with a large excess of tetranitromethane in a two-phase system. Modification of tyrosine to produce nitrotyrosine generates extrinsic Cotton effects near 410 nm in the visible region which are not affected by submicellar concentrations of taurodeoxycholate. On the other hand, supramicellar concentrations specifically altered the spectrum near 350 nm. The total reaction products included both inter- and intramolecular cross-links and the initial mixture can be separated into monomers and polymers (dimers). Despite the complicated reactions that have taken place, the monomer has maintained full activity in a tributyrin-taurodeoxycholate assay system and the evidence supports a more indirect role for tyrosine in colipase interface reactions.

Animals↗
PubMed1993

Labeling of tyrosines in proteins with [15N]tetranitromethane, a new NMR reporter for nitrotyrosines.

Lysozyme and ribonuclease were used as model proteins to explore the feasibility of detecting protein-bound nitrotyrosines by 15N-NMR spectroscopy. The reporter group was introduced via synthesized [15N]tetranitromethane. Several experiments for detection of the 15N resonance in the model [3-15N]nitrotyrosine demonstrated a substantial pH-dependence of the chemical shift. When lysozyme was nitrated, either two or three 15N resonances were detected, depending on the extent of nitration. The pH-dependence of the detected resonances clearly described an apparent microscopic pK in accord with reported values, while addition of Gd(III) gave selective line broadening, indicating that the 15N reporter group could also monitor relative distances from paramagnetic sources. Nitration of ribonuclease showed five 15N resonances, of which three persisted in the purified monomer. The pH-dependence of these resonances also described apparent microscopic pK values. The [3-15N]nitrotyrosine model was reduced to the [3-15N]aminotyrosine and its 15N resonance was easily monitored by several methods, including selective population inversion. When the protein-bound nitrotyrosines were similarly reduced, much sample decomposition resulted, a possible result of photooxidation, and/or reduction of disulfide bond(s), thereby making interpretation difficult.

Animals↗
PubMed1995

Modification with tetranitromethane of an essential tyrosine residue in uridine phosphorylase from Escherichia coli.

Treatment with tetranitromethane (TNM) rapidly and irreversibly inactivates uridine phosphorylase (UPase) from E. coli under mildly alkaline conditions. Modification of one of the four tyrosine residues decreases enzyme activity to 10%, while modification of all tyrosines decreases it to 8%. The second-order rate constant for the inactivation is 1250 +/- 50 M-1 min-1 at pH 8.0. Phosphate (0.1 M) does not affect the inactivation rate, while 5 mM uridine, or uridine plus phosphate nearly completely protect the enzyme against inactivation. Free sulfhydryl groups of UPase are not oxidized by TNM. A single modified peptide was isolated from tryptic digest by reverse-phase HPLC. The mass to charge ratio and the sequence determined are consisted with modification of Tyr-169, which corresponds to tryptic peptide 169Tyr-Asp-Thr-Tyr-Ser-Gly-Arg175. Tyrosine nitration leads to a significant decrease in the pKa of the phenolic hydroxy group without significantly affecting enzyme structure. Comparison of the pH dependence of activity and inactivation by diethylpyrocarbonate for the native and modified UPase reveals interaction between the modified tyrosine residue and an essential histidine residue (Drabikowska, A.K. and Wozniak, G (1990) Biochem. J. 270, 319-323). It is suggested that Tyr-169 takes part in the stabilization of the imidazole ring of the essential histidine in UPase.

Amino Acid Sequence↗
PubMed1996

Involvement of tyrosine residue in the inhibition of plant vacuolar H(+)-pyrophosphatase by tetranitromethane.

Plant vacuolar vesicles contain a novel H(+)-translocating pyrophosphatase (H(+)-PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H(+)-PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H(+)-translocating pyrophosphatase activity. The half-maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H(+)-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H(+)-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H(+)-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant (k2) of 0.039 s(-1) and a steady-state dissociation constant of inactivation (Ki) of 0.02 mM. Covalent modification of vacuolar H(+)-PPase by TNM increased Km value of the enzyme for its substrate without a significant effect on Vmax. Double logarithmic plots of the pseudo-first order rate constant (kobs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H(+)-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H(+)-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H(+)-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H(+)-PPase. Taken together, we suggest that vacuolar H(+)-pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM.

Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Modification of human transcortin by tetranitromethane. Evidence for the implication of a tyrosine residue in cortisol binding.

The effect of tetranitromethane on the cortisol binding activity of human transcortin has been investigated. This reagent induced a decrease of activity concomitant with nitration of tyrosine residues. An oxidation of sulphydryl groups was also observed but had no implication on cortisol binding. The nitration was specifically oriented in the site at pH6 and with low concentrations of reagent; under these conditions, a single essential tyrosine per molecule of transcortin seems implicated in cortisol binding. The absence of denaturation in modified transcortin was checked by circular dichroism spectra and polyacrylamide gel electrophoresis. Site specificity was demonstrated by full protection with cortisol against inactivation.

Binding Sites↗