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PubMed1995

Oxidized high-density lipoproteins modulate endothelin secretion by adult bovine aortic endothelial cells.

BACKGROUND: High-density lipoprotein (HDL) cholesterol levels are well established as an inverse risk factor for atherosclerosis. This fact is probably related to the ability of HDL to induce cholesterol efflux from the vascular cell. It is also possible that HDL affects the production of different mediators implicated in the development of atheroslerosis. Endothelin is a vasconstricting mitogenic peptide involved in the development of atherosclerosis. We studied whether native HDL, oxidized HDL and tetranitromethane HDL modulate the endothelin secretion of cultured adult bovine aortic endothelial cells. METHODS: We determined the effect of native HDL and modified HDLs (oxidized HDL and tetranitromethane HDL) on the secretion of endothelin by cultured adult bovine aortic endothelial cells. An endothelin radioimmunoassay system was used to quantify levels of immunoreactive endothelin in the cultured media. RESULTS: Native HDL, tetranitromethane HDL and oxidized HDL produced a highly significant stimulation of endothelin secretion (maximum 294% of control), even at low concentrations (10 and 20 micrograms/ml). Oxidized HDL2 and oxidized HDL3 produced a biphasic effect, with maximum secretion occurring with 100 micrograms/ml oxidized HDL3 (294% of control) and 50 micrograms/ml oxidized HDL2 (252% of control). The secretion of the peptide decreased with higher concentrations of oxidized HDL2 and oxidized HDL3. CONCLUSION: Because modified HDLs (oxidized HDL and tetranitromethane HDL) do not bind to the 'HDL receptor' to stimulate endothelin secretion, we propose that the stimulation of secretion is mediated by unspecific binding of the lipoprotein to the cell membrane. Nevertheless, oxidized HDL and tetranitromethane HDL may stimulate endothelin secretion via the scavenger-receptor pathway. Our results suggest that HDL and modified HDL participate in the regulation of vascular tone.

Animals↗

PubMed1984

On the mechanism of H+ translocation by mitochondrial H+ -ATPase. Studies with chemical modifier of tyrosine residues.

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.

Adenosine Triphosphate↗

PubMed1978

Effect of hydrolytic enzymes and protein-modifying reagents on gonadotropin receptors in bovine corpus luteum cell membranes.

Preincubation of membranes with various concentrations of pronase, trypsin, lipase, phospholipase A from Vipera russelli and from Crotalus durissus terrificus, phospholipase C from Bacillus cereus and from Clostridium welchii, acetic anhydride, 2,4-dinitrofluorobenzene and tetranitromethane resulted in a dose-dependent inhibition of 125I-labeled human choriogonadotropin binding. At the submaximal concentrations of enzymes and at both submaximal and maximal concentrations of protein-modifying reagents, the losses were always greater with 125I-labeled human choriogonadotropin than with 125I-labeled human lutropin. The inhibition of binding was a consequence of changes in the membranes rather than changes in the hormone caused by the agents being carried over to the final incubation. Inhibition of binding was non-competitive and irreversible. In untreated membranes, the 125I-labeled human choriogonadotropin binding was homogeneous (Kd = 1.7.10(-10) M; N = 60 fmol/mg protein). Treatment of membranes with various enzymes and protein-modifying reagents except tetranitromethane resulted in heterogeneous binding. The number of available high affinity receptors was greatly reduced in every case. However, the affinity of these sites were either unchanged (trypsin, lipase, phospholipase A from V. russelli, dinitrofluorobenzene and the tetranitromethane) or decreased (pronase and acetic anhydride). The newly appeared second receptor site had a Kd which varied from 3.2.10(-10) to 7.1.10(-9) M depending on the agent used, and the receptor numbers were low in all cases except acetic anhydride. Receptor occupancy conferred the receptors with marked protection against various hydrolytic enzymes, dinitrofluorobenzene and tetranitromethane. These data suggest that inhibition of binding by the above agents was primarily a consequence of changes in the receptor molecules themselves.

Animals↗

PubMed1983

Evidence for a carbanion intermediate in catalysis by spinach ribulose-1,5-bisphosphate carboxylase/oxygenase.

Spinach ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) showed marked reactivity towards tetranitromethane in the presence of RuBP, when the enzyme was in the fully activated state. The inactivated enzyme did not catalyze substrate-dependent nitroform production. Nitroform production was proportional to the concentration of the enzyme and tetranitromethane at optimal substrate concentrations. The pH optimum for nitroform production was similar to that for carboxylation. The effects of bicarbonate, ribulose-1,5-bisphosphate, and temperature on partition of the intermediate between production of phosphoglycerate and nitroform indicate that both routes share a common intermediate. Data on stoichiometry of the reaction imply that the intermediate is not oxidized by tetranitromethane but instead is nitrated. The data are compatible with a carbanion intermediate in the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase.

Anions↗

PubMed1976

Prostaglandin F2alpha receptors in bovine corpus luteum cell membranes. Effect of enzymes and protein reagents.

Various enzymes and protein reagents inhibited [3H]prostaglandin F2alpha binding to bovine corpus luteum cell membranes. Studies were undertaken (a) to explore further on the dose response relationships with the above agents, (b) to investigate the mechanism of inhibition of binding with respect to receptor affinities and number and (c) to assess whether decreased binding reflected changes in receptors and/or other membrane components. Preincubation of membranes with phospholipase A, trypsin, pronase, lipase, tetranitromethane, dinitrofluorobenzene, acetic anhydride and N-ethylmaleimide resulted in moderate to drastic inhibitions of [3H]prostaglandin F2alpha binding. The dose-dependent inhibition of binding by enzymes, but not by protein reagents (except for N-ethylmaleimide), exhibited a biphasic pattern: at lower concentrations, the loss of binding was low and relatively plateaued, but at higher concentrations, the losses were dramatic. The drastic reduction in binding by trypsin was due to destruction rather than solubilization of receptors from membranes. Phospholipase A was intrinsically more effective than phospholipases C and Ca2+ was not required for its inhibition of [3H]prostaglandin F2alpha binding. Protein reagents inhibition of binding was differently influenced by added Ca2+ i.e., loss of binding increased with some (N-ethylmaleimide), decreased with others (tetranitromethane, dinitrofluorobenzene and azobenzene sulfenylbromide). These results are interpreted to indicate that Ca2+ induced conformational changes in membranes which may result in exposure of new groups and burying of already exposed modifiable groups. Treatment of membranes with trypsin and N-ethylmaleimide selectively abolished high affinity prostaglandin F2alpha receptors. The low affinity receptors were present but their numbers as well as their affinity were decreased. Lipase, phospholipase A, acetic anhydride, dinitrofluorobenzene and tetranitromethane appear to decrease binding by totally abolishing all prostaglandin F2alpha receptors or by severely reducing their affinities. The occupancy of receptors by prostaglandin F2alpha afforded considerable protection against trypsin, phospholipase A, lipase and dinitrofluorobenzene. These data indicated that the inhibition of binding by the above agents, at least in part, can be attributable to changes in receptor sites alone.

Anhydrides↗

PubMed1981

Iodination of Escherichia coli ribosomal protein L18 abolishes its 5 S RNA binding activity.

Iodination of Escherichia coli ribosomal protein L18 inactivated the 5 S RNA binding activity of the protein. Complete activity loss occurred at a 4-fold molar excess of iodine to L18. Tyrosine was found to be the reactive amino acid. L18, prebound to 5 S RNA, was inactivated at a much slower rate than unbound L18. Treatment of L18 with tetranitromethane also resulted in an inactivation of the protein. However, much larger amounts of tetranitromethane, compared to iodine, were necessary to achieve inactivation (50% activity loss at a 600-fold molar excess of tetranitromethane to L18).

Carrier Proteins↗

PubMed1989

Chemical modification of tyrosine residues in glyoxalase I from yeast and human erythrocytes.

1. Yeast glyoxalase I was inactivated by N-acetylimidazole and tetranitromethane, the latter process following pK app 7.31 and irreversibly producing a protein with a spectrum typical of 3-nitrotyrosine. 2. For yeast glyoxalase I, amino-acid analysis and protection studies with S-(p-bromobenzyl)glutathione, a competitive inhibitor, indicated two classes of tetranitromethane-reactive tyrosine residues, fast- and slow-reacting, with the latter class containing the crucial tyrosine(s). 3. Human erythrocyte glyoxalase I was inactivated by tetranitromethane in fast and slow processes, protection studies in this case indicating the important tyrosine(s) as fast-reacting.

Amino Acids↗

PubMed1986

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy.

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed1987

Chemical modification and cross-linking of neurophysin tyrosine-49.

Photoaffinity labeling of the single neurophysin tyrosine, Tyr-49, with Met-Tyr-azido-Phe amide has been reported to inhibit both neurophysin self-association and peptide binding. Accordingly, we investigated the functional consequences of modification, principally by tetranitromethane, of Tyr-49. Tetranitromethane-mediated tyrosine-tyrosine cross-linking permitted synthesis of covalent neurophysin "dimers" and of peptide-protein conjugates, the latter potentially analogous to the photoaffinity-labeled product. The self-association and binding properties of the covalent dimers were found to be similar or enhanced relative to those of the native protein. In contrast to the photoaffinity-labeled product, covalent conjugates of Tyr-49 with the ligand peptides Met-Phe-Tyr amide, Phe-Tyr amide, and Tyr-Phe amide also generally exhibited normal or increased binding affinity for exogenous peptide; a subfraction of the Phe-Tyr amide adducts showed evidence of reduced affinity. Diiodination of Tyr-49 had no significant effect on binding. However, among the products of tetranitromethane treatment in the absence of peptide was a novel inactive non-cross-linked product, representing modification only of Tyr-49 but containing no demonstrable nitrophenol. As evidenced by circular dichroism and nuclear magnetic resonance (NMR), this product was not significantly unfolded and retained the ability to self-associate. These latter results provide the strongest evidence thus far of a role for Tyr-49 in peptide-hormone binding. The disparate effects of different Tyr-49 modifications are collectively interpreted and reconciled with NMR data and the properties of the photoaffinity-labeled protein to suggest potential mechanisms of Tyr-49 participation in binding and the probable orientation of Tyr-49 relative to peptide residue 3 in neurophysin complexes.

Amino Acids↗

PubMed1989

Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q.

Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.

Amino Acid Sequence↗

PubMed1980

Tyrosine residues in the C-terminal domain of the elongation factor G are essential for its interaction with the ribosome.

Chemical modification of the elongation factor G (EF-G) with tetranitromethane and iodine has been studied. It has been shown by spectrophotometric titration that EF-G contains two exposed tyrosine residues, one of which has an unusually low pK value for a phenol hydroxyl group at pH 8.5. Modification of one tyrosine residue with either tetranitromethane or iodine results in a 70--80% loss of EF-G activity in all ribosome-dependent reactions. Modification of three or four residues inhibits 90--100% of activity. Binding of EF-G with the 70-S ribosome and 50-S subunit is equally effective for protection of tyrosine residues against modification. The rate of EF-G modification with tetranitromethane is considerably higher in the presence of guanyl nucleotides than for free EF-G. The modified residues are located in the C-terminal domain of EF-G and are presumably contained in one of the sites of EF-G interaction with the ribosome.

Escherichia coli↗

PubMed1989

Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site.

4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.

Affinity Labels↗

PubMed1981

Selective loss of elastase inhibitory activity of alpha 1-proteinase inhibitor upon chemical modification of its tyrosyl residues.

Nitration or acetylation of tyrosine residues of human plasma alpha 1-proteinase inhibitor (alpha 1-PI) with tetranitromethane or N-acetylimidazole resulted in a selective loss of its inhibitory activity. While the chymotrypsin- and trypsin-inhibitory activities were unaffected, all of the elastase inhibiting activity was lost. Comparative amino acid analyses of the native inhibitor and the chemically modified derivatives revealed no differences in the amino acid compositions with the exception that in the case of the tetranitromethane-treated material there were losses in the tyrosine content which could be accounted for by the formation of 3-nitrotyrosine. Nitration of alpha 1-PI.trypsin, alpha 1-PI.chymotrypsin, and alpha 1-PI.elastase complexes with excess tetranitromethane indicated that complex formation protected the binding site of the inhibitor for elastase, but not for trypsin or chymotrypsin. Quantitative estimation of the number of nitrotyrosine residues in the alpha 1-PI isolated from the complexes has shown that one tyrosine residue was protected from nitration. Competition studies have also indicated that the elastase inhibitory site is different from that of trypsin or chymotrypsin, although the possibility of overlapping sites could not be disregarded.

Acetylation↗

PubMed1989

Dissociation of various biological activities of Clostridium perfringens alpha toxin by chemical modification.

The effect of N-acetylimidazole, tetranitromethane, maleic anhydride and N-ethylmaleimide on various biological activities of Clostridium perfringens alpha (alpha)-toxin was investigated. Treatment of the toxin with N-acetylimidazole, tetranitromethane or maleic anhydride resulted in significant reduction of lethal, hemolytic and platelet-aggregating activities and phospholipase C activity (EY activity), as measured by increased turbidity in egg yolk emulsions. However, EY activity was more resistant to these reagents than lethal, hemolytic or aggregating activities. Phospholipase C activity (PN activity) as measured by hydrolysis of p-nitrophenylphosphorylcholine was retained after treatment with N-acetylimidazole, tetranitromethane or maleic anhydride. The activities of the toxin were not inactivated by treatment with N-ethylmaleimide. These data suggest that alpha-toxin contains multiple sites for biological activities of the toxin.

Animals↗

PubMed1995

Potentiation of retinoic acid-induced U-937 differentiation into respiratory burst-competent cells by nitric oxide donors.

All-trans retinoic acid (tretinoin) is a known inducer of differentiation of the human monoblastic cell line, U-937. We now report that the ability of retinoic acid (RA) to induce differentiation of U-937 cells into cells possessing respiratory burst activity is enhanced by the known nitric oxide-donating drugs glyceryl trinitrate, molsidomine and CAS 936, and by tetranitromethane in combination with cysteine. RA alone was a strong inducer of U-937 differentiation as indicated by the following responses to 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation: (1) increase in the percentage of cells staining with nitroblue tetrazolium (NBT); (2) increase in the total amount of formazan (the product of NBT reduction by O2-.) as determined spectrophotometrically; (3) increase in hexose monophosphate shunt (HMPS) activity as assessed by [14C]CO2 released from D-[1-14C]glucose. RA was also able to increase mRNA levels for two respiratory burst-related genes and for glucose-6-phosphate dehydrogenase (G6PD), an HMPS enzyme. Other indications of differentiation were reduced cell proliferation, increased adherence and altered nuclear morphology. The observed increase in formazan production and HMPS activity and the reduction of cell proliferation due to RA were augmented by co-treatment with either glyceryl trinitrate, molsidomine, CAS 936 or tetranitromethane plus cysteine. Glyceryl trinitrate alone increased HMPS activity and G6PD mRNA levels and also reduced cell proliferation. Glyceryl trinitrate, molsidomine and CAS 936 are presumed to release nitric oxide and increase intracellular cGMP levels by stimulation of soluble guanylate cyclase. The mechanism of action of tetranitromethane is less certain, although it may also generate reactive nitrogen intermediates. These data suggest that a NO./cGMP pathway may augment a retinoic acid-mediated pathway to enhance maturation of U-937 cells with respect to the respiratory burst. Glyceryl trinitrate may act additionally by another pathway.

Cell Differentiation↗

PubMed1996

Interactions of glycine and strychnine with their receptor recognition sites in mouse spinal cord.

Interactions between the inhibitory neurotransmitter glycine and its receptor antagonist strychnine have been studied in mouse spinal cord membranes and both agents employed to protect against residue selective protein modifying reagents in order to identify contact residues for ligand binding. Glycine was found to behave as a full competitive inhibitor of [3H]-strychnine binding, provided that precautions were taken to prevent radioligand binding to the glass-fibre filters used to terminate the assays. Hill coefficients for the glycine inhibition of [3H]-strychnine binding were not significantly different from one, indicating a lack of cooperative interactions. For the protection experiments, N-bromosuccinimide, tetranitromethane, diethylpyrocarbonate and 2,3-butanedione were used under conditions selective for tryptophan, tyrosine, histidine and arginine residues, respectively. Of these reagents, N-bromosuccinimide, tetranitromethane and diethylpyrocarbonate caused a decrease in total [3H]-strychnine binding without affecting the ability of unlabelled strychnine to compete. In contrast, the same reagents disrupted the ability of glycine to inhibit [3H]-strychnine binding. The presence of either excess glycine (10(-2) M) or strychnine (10(-4) M) during the above treatments was found to prevent the decrease in total and strychnine-specific [3H]-strychnine binding. However, only in the case of diethylpyrocarbonate treatment were both agonist and antagonist able to protect against the loss of glycine-specific [3H]-strychnine binding. The reagent 2,3-butanedione caused an increase in total and strychnine-specific [3H]-strychnine binding (which we have shown elsewhere to be at a site unrelated to the inhibitory glycine receptor). When the above protein modifying reagents were applied under the same conditions to specific strychnine binding antibodies, all four caused significant decreases in subsequent [3H]-strychnine binding. Strychnine was found to afford significant protection of the antibodies against N-bromosuccinimide, tetranitromethane and 2,3-butanedione, but not against diethylpyrocarbonate. Our results suggest that glycine and strychnine compete at overlapping but conformationally distinct sites on the receptor. Tyrosine, tryptophan, histidine and arginine residues are implicated as strychnine contact residues with a shared role for histidine in the recognition of glycine.

Animals↗

PubMed1999

Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous.

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.

Amino Acid Sequence↗

PubMed1993

Evidence for alpha-proton abstraction and carbanion formation involving a functional histidine residue in lentil seedling amine oxidase.

Lentil seedling amine oxidase catalyzes the oxidation of putrescine and in the presence of tetranitromethane gives rise to the formation of nitroform anion. The initial rate of substrate and enzyme-dependent nitroform production is linearly related to the functional active site content and is proportional to the tetranitromethane concentration. Diethylpyrocarbonate modifies two histidyl residues on the lentil amine oxidase. Incubation of the enzyme with diethylpyrocarbonate at 25 degrees C and pH 7.0 irreversibly inhibits enzyme activity by a pseudo first-order kinetics process. The data obtained are consistent with the enzyme-dependent abstraction of an alpha-proton from the substrate to form an intermediate enzyme bound carbanion and indicate a functional role for histidine in lentil amine oxidase catalysis consistent with that of a general base in proton abstraction.

Amine Oxidase (Copper-Containing)↗