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.

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.

Mavicyanin, a stellacyanin-like protein from zucchini peelings: primary structure and comparison with other cupredoxins.

The complete amino-acid sequence of mavicyanin, a small blue copper-containing glycoprotein isolated from zucchini peelings, is presented. The sequence of this cupredoxin was deduced from analysis of peptides obtained after cleavage of the protein with trypsin or Asp-N endoproteinase. Mavicyanin consists of a single polypeptide chain of 108 amino-acid residues. Accurate molecular weight determination by electrospray mass spectrometry (12 752 Da) indicates a mass difference of approx. 1005 Da with respect to the mass of the protein, as determined on the basis of the amino-acid sequence (11747 Da). This difference was tentatively assigned to the carbohydrate moiety, not yet characterized, attached to the protein via an N-linkage to Asn-58 and O-linkages to unidentified Ser/Thr residues. The comparison of the primary structure of mavicyanin with those of other cupredoxins shows that three copper ligands (His-44, Cys-57 and His-90) are conserved, while a glutamine residue (Gln-95), as in stellacyanin, is possibly the fourth ligand. An amino-acid sequence alignment of mavicyanin with copper proteins currently identified as phytocyanins is also proposed, showing same invariant residues in key positions related to the maintenance of the beta-barrel fold and to the active site.

The interaction of nitric oxide with ascorbate oxidase.

1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.

X-ray absorption edge spectroscopy of Co(II)-binding sites of copper- and zinc-containing proteins.

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and [...-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase greater than stellacyanin greater than superoxide dismutase greater than or equal to carbonic anhydrase.

Enhancement of daunomycin toxicity by the differentiation inducer hexamethylene bisacetamide in erythroleukemia cells.

Cytotoxic effects of daunomycin were investigated upon differentiation of Friend erythroleukemia cells induced with hexamethylene bisacetamide, a process during which a 20-fold increase in the hemoglobin content occurred. Daunomycin proved to be more toxic to differentiated Friend cells than to their undifferentiated counterparts. No changes in the daunomycin uptake rates of the two cell types were detectable. Externally added catalase and desferrioxamine mesylate protected against the additional cytotoxicity of daunomycin in differentiated cells, pointing to hydrogen peroxide and iron ions as mediators of the toxic effect. Daunomycin-dependent, cyanide-insensitive oxygen consumption of control and induced cells did not differ significantly, and the rate of formation of the daunomycin semiquinone radical electron paramagnetic resonance signal was similar in both cell types, indicating that the difference in toxicity was not due to increased drug activation by plasma membrane enzymes. Differentiated cells had a lowered catalase content; the cellular iron content was shown to increase by 2.8-fold upon cell differentiation, with hemoglobin-bound iron being about 50% of the total. Altogether, the results suggest increased intracellular hydrogen peroxide generation mediated by hemoglobin, combined with a decrease in catalase activity and an increase in accessible iron, as responsible for the higher sensitivity to daunomycin shown by differentiated Friend cells. This represents the first experimental system where the increase in anthracycline cytotoxicity upon cell differentiation can be attributed to redox activation and the formation of reactive oxygen species.

Status of the cofactor identity in copper oxidative enzymes.

Much conflicting data have appeared in the literature regarding the nature of the active site structures responsible for catalysis in three classes of copper enzymes: the copper amine oxidases, dopamine beta-monooxygenase and galactose oxidase. Although pyrroloquinoline quinone has been proposed to be the active site cofactor in each instance, new findings indicate this is not the case. Instead, recently available data indicate a spectrum of strategies for substrate activation, which range from direct metal catalysis (dopamine beta-monooxygenase) to the involvement of protein-derived radicals (galactose oxidase) and protein-derived quinones (copper amine oxidases).

Protection against apoptosis by monoamine oxidase A inhibitors.

Several lines of evidence have been accumulating indicating that an important role may be played by mitochondrial homeostasis in the initiation phase, the first stage of apoptosis. This work describes the results obtained by using different inhibitors of monoamine oxidases (MAO), i.e. pargyline, clorgyline and deprenyl, on mitochondrial integrity and apoptosis. Both pargyline and clorgyline are capable of protecting cells from apoptosis induced by serum starvation while deprenyl is ineffective. These data represent the first demonstration that MAO-A inhibitors may protect cells from apoptosis through a mechanism involving the maintenance of mitochondrial homeostasis.

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.

Inhibition of copper-dependent amine oxidases by some hydrazides of pyrrol-1-ylbenzoic and pyrrol-1-ylphenylacetic acids.

Some hydrazides of pyrrol-1-ylbenzoic and pyrrol-1-ylphenylacetic acids were prepared, and their effect on copper-dependent amine oxidases (Cu-AOs) and FAD monoamine oxidases (MAOs) activities was tested. The compounds were not substrates for Cu-AO enzymes but acted as noncompetitive inhibitors. Hydrazides of pyrrol-1-ylphenylacetic acids were highly specific for plasma amine oxidase (Ki = 0.5-1 microM). In contrast, all the hydrazides were weak inhibitors of MAO activity. Incubation with the hydrazide derivatives led to irreversible inactivation of Cu-AOs. Therefore, the inhibition implied two distinct steps. The first one consisted of the rapid formation of the enzyme-inhibitor complex and was reversed by dialysis. In the second step, the complex was irreversibly transformed, probably by the formation of a Schiff base between the hydrazide and the prosthetic carbonyl group of the enzyme.

Heat enhancement of cytotoxicity induced by oxidation products of spermine in Chinese hamster ovary cells.

This study investigates the potential of using polyamines as thermosensitizers, in the presence of bovine serum amine oxidase (BSAO), as a new anticancer strategy. The effect of hyperthermia on cytotoxicity of spermine oxidized by purified bovine serum amine oxidase was investigated in Chinese hamster ovary cells. Several different spermine concentrations were employed in the presence of BSAO at 37 degrees and 42 degrees. Cytotoxicity was considerably enhanced at 42 degrees. Heat also increased the individual cytotoxicity of both exogenous H2O2 and the exogenous aldehyde acrolein. Thus, both of these species could contribute to the thermal enhancement of cytotoxicity caused by BSAO and spermine. The effect of temperature was especially marked in the presence of exogenous catalase. This cytotoxicity cannot be accounted for by H2O2 and was attributed to aldehyde(s). The involvement of aldehyde(s) in cytotoxicity at 42 degrees was also confirmed by the complete inhibition of cytotoxicity with both exogenous aldehyde dehydrogenase and exogenous catalase. A particularly interesting finding, in the presence of exogenous catalase, was that conditions of BSAO and spermine (< or = 50 microM) which were non-toxic at 37 degrees became cytotoxic at 42 degrees. This suggests that spermine-derived aldehyde(s), that were non-toxic at 37 degrees, contributed to cytotoxicity at 42 degrees and resemble thermosensitizers. The thermosensitizing activity of aldehyde(s) produced in the BSAO-catalysed oxidation of spermine has potential value for improving the therapeutic effects of hyperthermia and could be considered for future application in cancer therapy. Polyamines are present at elevated levels in tumour cells and have been considered as heat sensitizers. By delivering BSAO into tumour cells, toxic oxidation products of polyamines could be produced in situ for selective killing of tumour cells.

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.

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.

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.