Inducible expression of maize polyamine oxidase in the nucleus of MCF-7 human breast cancer cells confers sensitivity to etoposide.

In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H(2)O(2), has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine.

Substrate specificity of copper-containing plant amine oxidases.

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.

Interaction of biologically active amines with mitochondria and their role in the mitochondrial-mediated pathway of apoptosis.

The natural polyamines spermine, spermidine and putrescine, polycationic molecules at physiological pH, interact with mitochondrial membranes at two specific binding sites exhibiting low affinity and high binding capacity. This binding represents the first step in the electrophoretic mechanism of polyamine transport into mitochondria. Spermine accumulated into the mitochondrial matrix is able to flow out by an electroneutral mechanism. This process promotes bi-directional transport of polyamines in and out of mitochondria, driven by electrical potential and pH gradient, respectively. Polyamines and biogenic amines are oxidized by cytosolic and mitochondrial amine oxidases with the production of hydrogen peroxide and aldehydes, both of which are involved in the induction and/or amplification of the mitochondrial permeability transition (MPT). This phenomenon, which provokes a bioenergetic collapse and redox catastrophe, is strongly inhibited by polyamines in isolated mitochondria. Monoamines also exhibit an inhibitory effect at higher concentrations, but at low concentrations behave as inducer agents. MPT is characterized by the opening of a channel, the transition pore, which permits non-specific bi-directional traffic of solutes across the inner membrane, leading to swelling of the organelle and release of cytochrome c and apoptosis-inducing factors. These proteins in turn activate the caspase-cascade, which triggers the apoptotic pathway. Depending on their cytosolic concentration, metabolic conditions and cell type, polyamines act as promoting, modulating or protective agents in mitochondrial-mediated apoptosis. While their protective effect could reflect inhibition of MPT and retention of cytochrome c, the promoting effect can be explained by the generation of reactive oxygen species that induce the opposite effect on MPT and cytochrome c release. Polyamines and other active amines can also participate in the regulation of apoptotic pathways by interacting with the mitochondrial tyrosine phosphorylation/dephosphorylation system. Future studies of the multifaceted interactions of polyamines with mitochondria will thus have a substantial impact on our understanding of the physiology of cell proliferation death at several mechanistic levels.

Biogenic amines and apoptosis: minireview article.

The programmed cell death is a very complex mechanism involving many factors, among them the intracellular concentration of biogenic amines (BA) appears to be important for apoptosis triggering. The mitochondrial damage is imputable to hydrogen peroxide and aldehydes, produced by amine oxidases (AO)-mediated oxidation of BA. On the other hands, the apoptosis protection observed by high BA concentration appears to be related to their scavenger effect of ROS and/or their interaction with membrane pores. Also monoamine oxidase (MAO) inhibitors, like propargylamines, preserve the mitochondria integrity by inhibiting MAO and therefore the production of H2O2 and aldehydes and, as cations, by regulating membrane pores, like BA. As general conclusion, apoptosis is protected by high concentration of BA and/or other cations while it is favoured by ROS produced by AOs or other mechanisms.

Amine oxidases and tumors.

The physiological function in living organisms of amine oxidases, is not completely established but is certainly related to the biogenic amines metabolism and therefore involved in essential processes such as the cell growth and differentiation. A correlation between degree of tumor malignancy and level of AO activity has been reported. The catalytic products of oxidative deamination of amines (hydrogen peroxide and aminoaldehyde) exert a cytotoxic effect and are considered cell growth inhibitors. The biogenic amines in same way could be considered in the cells as both poisons and protectors. A balance of oxidant and antioxidant enzymes appears to be very important in carcinogenesis and cell growth regulation.

Is the catalytic mechanism of bacteria, plant, and mammal copper-TPQ amine oxidases identical?

This short review is mostly concerned with the work carried out in Rome on the copper amine oxidase from bovine serum (BSAO). The first target was the copper oxidation state and its relationship with the organic cofactor. It was found that copper is not reduced on reaction with amines under anaerobic conditions or along the catalytic cycle and that it is not within bonding distance of the quinone cofactor. The copper stability in the oxidised state was supported by BSAO ability to oxidise benzylhydrazine, a slow substrate, in the presence of N,N-diethyldithiocarbamate (DDC) and by the substantial catalytic activity of Co(2+)-substituted BSAO. Parallel work established that only one subunit of the dimeric enzyme readily binds reagents of the carbonyl group. Flexible hydrazides with a long aromatic tail were found to be highly specific inhibitors, suggesting the presence of an extended hydrophobic region at the catalytic site. A study by stopped-flow transient spectroscopy and steady state kinetics led to the formulation of a simplified, yet complete and consistent, catalytic mechanism for BSAO that was compared with that available for lentil seedling amine oxidase (LSAO). As in other copper amine oxidases, BSAO is inactivated by H(2)O(2) produced in the catalytic reaction, while the cofactor is stabilised in its reduced state. A conserved tyrosine hydrogen-bonded to the cofactor might be oxidised.

New perspectives on the role of amine oxidases in physiopathology.

In the paper here presented we summarize some results obtained in our laboratory in the last few years on new structural and functional aspects of some amine oxidases (AOs), which have to be taken into consideration in defining new strategies of controlling the cellular physiopathology. In particular, the ability of Cu-AO purified from vegetal sources or from bovine serum to bind different cellular targets inducing in them conformational as well as chemical modifications are described and the consequences of this interaction on cellular functions are discussed. This is the case of the protective effect of Cu-AO against the damage induced by free radicals, cell enrichment with Cu-AO, induction of cataract and the leukocyte-endothelia interaction. The role of Cu and FAD-amine oxidases related as to the protection or damage of cells is also discussed. In this context the involvement of MAOs in the modulation of the mitochondrial functions and in the induction of apoptosis is described and some aspects of the molecular mechanism of AO inhibition by H(2)O(2) and metronidazole analyzed.

Some new functions of amine oxidases.

Two contrasting topics are examined in this account: the protective actions of amine oxidases (AOs) resulting from the elimination and/or modulation of the levels of polyamines and some biogenic amines, such as histamine, in anaphylactic shock and the cell damaging effect of AOs catabolic products. Other functions of the plasma copper-containing amine oxidase are considered; namely the modification of some proteins by oxidation of their free amino groups, the auto-regulation of the catalytic activity of AOs, the protective effect against free radicals, and the regulation of K(+)-channels.

Tyramine and monoamine oxidase inhibitors as modulators of the mitochondrial membrane permeability transition.

Incubation of rat liver mitochondria with 100-500 mM tyramine, a substrate for monoamine oxidases A and B (MAOs), in the presence of 30 mM Ca2+ induces matrix swelling, accompanied by collapse of membrane potential, efflux of endogenous Mg2+ and accumulated Ca2+ and oxidation of endogenous pyridine nucleotides. These effects are completely abolished in the presence of cyclosporin A, ADP, dithioerythritol and N-ethylmaleimide, thus confirming the induction of the mitochondrial membrane permeability transition (MPT). The observed partial protective effect exerted by catalase indicates the involvement of both MAO-derived hydrogen peroxide and aldehyde. Higher concentrations of tyramine (1-2 mM) are less effective or even completely ineffective. At these high concentrations tyramine has an inhibitory effect when the MPT is induced by 100 mM Ca2+. The MAO inhibitors clorgyline (50 mM) and pargyline (500 mM) completely protect against MPT induction by 100 mM tyramine but also inhibit the phenomenon, although with different efficacy, when it is induced by 100 mM Ca2+ in the absence of tyramine. Taken together, our data suggest that tyramine, clorgyline and pargyline act as modulators of the MPT either through a direct inducing/protective effect or by controlling hydrogen peroxide and aldehyde generation.

Redox properties and acid-base equilibria of zucchini mavicyanin.

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.

The oxidation and reduction reactions of bovine serum amine oxidase. A kinetic study.

The presteady-state and steady-state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped-flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady-state parameters. The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. & Klinman, J.P. (1998) Biochemistry 37, 12513-12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured. The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate. These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans-iminative proton abstraction occurring in the enzyme-substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine-radical intermediate, while the latter is less easily interpreted.

Modulation of bovine serum amine oxidase activity by hydrogen peroxide.

Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.

The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors.

Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic co-factor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm. The band shifts to 526 nm around 60 degrees C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu2+ depletion. Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystalline Escherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure. The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution. The structural stability of the adduct in the absence of Cu2+ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct. Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct. On the basis of this model and of the catalytic competence of Co(2+)-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed.

Serum amine oxidase can specifically recognize and oxidize aminohexyl (AH) chains on AH-Sepharose support: single-step affinity immobilization.

Preparative affinity chromatography of bovine serum amine oxidase (SAO) on aminohexyl (AH)-Sepharose was often associated with an unexpected irreversible SAO retention on the support. This particular enzyme immobilization, occurring without coupling reagents, was supposed to be due to a SAO ability to: (i) recognize alkylamine groups of the support as macro-molecularized substrate; (ii) catalyse their oxidation to the corresponding aldehydes, with release of NH3 and H2O2; and (iii) be immobilized on the activated support by a coupling between the nascent aldehyde groups and SAO free amine groups. This affinity immobilization procedure, with the self-activation of the support, being mild, allows by simple incubation for 24 h, the enzyme immobilization with the retention of 80% from original specific activity of free SAO. Immobilized SAO on AH-Sepharose microcolumns, viewed as a continuous flow-system reactor, was able to catalyse benzylamine oxidation for several weeks.

Characterization of the haemoglobin-mediated inhibition of the enzymatic activity of bovine serum amine oxidase.

Haemoglobin has been previously identified as responsible for the decreased enzymatic activity of copper bovine serum amine oxidase (BSAO) in suspensions of human or bovine hemolyzed erythrocytes [Marcocci, L., Pietrangeli, P., Befani, O., Mavelli, I., & Mondovi', B. (1991b) Life Chem. Report, 9, 171-177]. This is confirmed by present results on bovine methaemoglobin. Bovine globin and horse skeletal muscle mioglobin showed a similar inhibiting ability, but neither bovine serum albumin nor cytochrome c inhibited BSAO activity under the same experimental conditions. The inhibitory effect of bovine haemoglobin was dependent on pH only at high buffer ionic strength. It was highest in physiological conditions (PBS) where haemoglobin acted as a reversible non competitive inhibitor of BSAO activity, with apparent Ki of 0.5 mM at 37 degrees C. The inhibition was unaffected by partial BSAO deglycosylation (40% of glucidic residues removed) but decreased when haemoglobin lysine groups were derivatised using citraconic anhydride. A possible molecular mechanism underlying the inhibitory effect is discussed.