Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach.

The polyamines spermine, spermidine and putrescine are ubiquitous cell components. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper-containing amine oxidases. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. In tumors, polyamines and amine oxidases are increased as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H(2)O(2) and aldehydes produced by the reaction. This study demonstrated that multidrug-resistant (MDR) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding wild-type (WT) ones to H(2)O(2) and aldehydes, the products of BSAO-catalyzed oxidation of spermine. Transmission electron microscopy (TEM) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. Increasing the incubation temperature from 37 to 42 degrees Celsius enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumor growth, particularly well if the enzyme has been conjugated to a biocompatible hydrogel polymers. Since both wild-type and MDR cancer cells after pre-treatment with MDL 72527, a lysosomotropic compound, are sensitized to subsequent exposure to BSAO/spermine, it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells. It is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.

Polyketone polymer: a new support for direct enzyme immobilization.

Polyketone polymer -[-CO-CH(2)-CH(2)-](n)-, obtained by copolymerization of ethene and carbon monoxide, is utilized for immobilization of three different enzymes, one peroxidase from horseradish (HRP) and two amine oxidases, from bovine serum (BSAO) and lentil seedlings (LSAO). The easy immobilization procedure is carried out in diluted buffer, at pH 7.0 and 3 degrees C, gently mixing the proteins with the polymer. No bifunctional reagents and spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain. Experiments demonstrate a high linking capacity of polymer for BSAO and an extraordinary strong linkage for LSAO. Moreover, activity measurements demonstrate that immobilized LSAO totally retains the catalytic characteristics of the free enzyme, where only a limited increase of K(M) value is observed. Finally, the HRP-activated polymer is successfully used as active packed bed of an enzymatic reactor for continuous flow conversion and flow injection analysis of hydrogen peroxide containing solutions.

EPR study of spermine interaction with multilamellar phosphatidylcholine liposomes.

The interaction of spermine with egg-yolk phosphatidylcholine liposomes was investigated. The EPR spin labeling technique evidenced that spermine induces modifications of some membrane functions of biological interest like water permeability and is a possible modulator of diffusion processes for charged and polar molecules. The association constant for a hypothesized complex between spermine and the phosphate group of phosphatidylcholine was evaluated by enzymatic methods.

Effect of polyphosphates on the activity of amine oxidases.

The interaction between polyphosphates and polyamines was investigated by 31P-NMR spectroscopy and by amine oxidase activity measurements. An apparent competition between negatively charged polyphosphates (ATP, ADP, AMP, tripolyphosphate and pyrophosphate) and positively charged polyamine, for the active site of bovine serum and soybean seedling amine oxidases, was observed by activity measurements. This behavior was explained by formation of polyamine-polyphosphate complexes and the stability constants of these complexes were calculated by 31P NMR. However, at a given concentration of polyphosphate, the amine oxidase activity was found higher than that expected on the basis of the free amine concentration calculated according to the NMR stability constant. This fact, and the different extent of inhibition of the spermidine oxidase activity of soybean seedling and of bovine serum amine oxidases observed in the presence of a given polyphosphate, suggest that amine oxidases may be active also on the polyamine-polyphosphate complexes. This hypothesis was supported by the strong dependence of the kcat/Km of bovine serum amine oxidase on ionic strength, indicating an electrostatic interaction between the charged amine and the active site, while no effect of ionic strength on kcat/Km was observed in the presence of ATP. A kinetic model of this behavior was found to fit the experimental data.

Interaction of spermine with dimyristoyl-L-alpha-phosphatidyl-DL-glycerol multilamellar liposomes.

Polycationic spermine interacts with the negative phosphate group of dimyristoylphosphatidylglycerol multilamellar liposomes, forming a positively charged shell around the vesicle surface. An association constant of (2.15+/-0.45) x 10(3) M(-1) between spermine and the phospholipid groups in liposomes has been evaluated by a new and rapid enzymatic method. ESR spectra show that the effects of this polycation on liposomes are substantially different from those of cations like Ca2+ and Mg2+ and confirm the ability of spermine to induce liposome aggregation and not fusion.

Interaction of linear mono- and diamines with dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol multilamellar liposomes.

The effect of linear monoamines on dimyristoylphosphatidylglycerol and dimyristoylphosphatidylcholine multilamellar liposomes was studied as a function of their length and compared with the behavior of linear carboxylic acids. The role of the hydrophobic interactions was demonstrated and the free energy of the binding for each interacting carbon atom was determined. The thermotropic behavior of the liposomes was characterized by differential scanning calorimetry and it was shown that these molecules affect the temperature and the cooperativity of the gel to fluid state transition of the membrane differently. In particular, it appeared that membrane perturbation was maximum when the chain length of the amphipathic molecules ranged between 7 and 9 carbon atoms, with more pronounced effects in the case of monoamines. Molecules shorter than 3-4 carbon atoms did not produce any observable change in the transition temperature. The study was extended to linear alpha,omega-diamines to investigate the amphipathic character of long diamines and to investigate the role of bridging bonds established with neighboring phospholipids.

Thermodynamic analysis of the oxidative deamination of polyamines by bovine serum amine oxidase.

The effect of incubation temperature on the activity of bovine serum amine oxidase was studied using natural substrates of this enzyme, namely spermine and spermidine. The activity behavior was found to be rather complex and different from that observed using benzylamine as substrate. The enzyme is fully active after 3 min incubation at temperatures up to 60 degrees C, while at higher temperatures it shows an S-shaped irreversible decrease of the activity with a T50 of about 70 degrees C. The dependence of the kinetic parameters Kcat and Km on temperature was also studied in the range 5-66 degrees C, measuring the initial rates of oxidation of spermidine. The Arrhenius plot of kcat shows a continuous bend in the thermal stability range of the enzyme. This nonlinearity is due to the dramatic change of kcat activation entropy (from +11 eu at 8 degrees C, to -31 eu at 50 degrees C) and indicates that kcat is a composite constant, involving two or more successive steps characterized by different kinetic parameters. In the case of the ratio kcat/Km, which for natural substrates of bovine serum amine oxidase was shown that its dependence on ionic strength gives information on the influence of electrostatic field on k1 (Stevanato et al. (1994) Biochem. J. 299, 317-320), where k1 is the kinetic constant of interaction between the polyamine and the enzyme, the Arrhenius plot is linear in the range 12-45 degrees C, and an activation entropy of 18.8 eu was calculated at 37 degrees C. This value is in accord with a mechanism involving the formation of a polyamine-enzyme complex which is accompanied by neutralization of charges between the polycationic substrate and negatively charged groups of the enzyme.

Kinetics and free energy profiles of spermine transport in liver mitochondria.

In the present study, the voltage-dependent mechanism of spermine transport in liver mitochondria [Toninello, A., Dalla Via, L., Siliprandi, D., and Garlid, K. D. (1992) J. Biol. Chem. 267, 18393-18397] was further characterized by determining the rate constants J(max) and K(m) as functions of membrane potential. An increase in mitochondrial membrane potential from 150 to 210 mV promoted spermine transport, as reflected by an approximate 4-fold increase in J(max) and 25% decrease in K(m). The mechanism for the voltage dependence of transport was examined using the beta value, i. e., the slope of ln(flux) vs FDeltaPsi/RT plots. Flux-voltage analyses performed at very high and very low spermine concentrations yielded beta values of 0.125 and 0.25, for J(max) and J(max)/K(m), respectively. The physical significance of these beta values was analyzed by means of a theory relating the enzyme reaction rate to the free energy profiles [Yagisawa, S. (1985) Biochem. J. 303, 305-311]. Depending on the nature of K(m), two possible models could be proposed to describe the location and shape of the barriers in the membrane. Analysis of previous data concerning spermine binding [Dalla Via, L., Di Noto, V., Siliprandi, D., and Toninello, A. (1996) Biochim. Biophys. Acta 1284, 247-252] by a new rationale provided evidence for an asymmetrical energy profile composed of two peaks with the binding site near the membrane surface followed by a rate-determining energy barrier for the movement of the bound spermine toward the internal region of the membrane.

Superoxide ion as active intermediate in the autoxidation of ascorbate by molecular oxygen. Effect of superoxide dismutase.

The oxidation of ascorbic acid by molecular oxygen was investigated by optical and magnetic resonance methods in the presence and in the absence of copper and iron complexes. The results show that one superoxide ion is generated for each oxidized molecule of ascorbate. The effect of addition of Cu,Zn superoxide dismutase, which at concentrations higher than 10(-7) M halves the oxidation rate, is discussed and a reaction mechanism is proposed. An upper limit for the kinetic rate constant of the reaction between the ascorbate radical and the superoxide ion is reported.

Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase.

Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide. The properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxidase. The immobilized enzyme was utilized in a flow-through system equipped with a polarographic detector which monitors the oxygen depletion due to the reaction ascorbic acid + 1/2 O2----dehydroascorbic acid + H2O. This method, the response of which is linear between 3 X 10(-7) and 5 X 10(-4) M ascorbate, was utilized to measure the ascorbic acid in biological samples such as human plasma and fruit juices at a rate of about 60 determinations every hour with a standard deviation lower than 5%.

The active site of manganese-containing superoxide dismutase from Bacillus stearothermophilus studied by 1H and 19F magnetic relaxation.

The dependence of the magnetic relaxation rates of 1H and 19F- on temperature, frequency, pH and N-3 concentration, were measured in solutions of Manganese-containing superoxide dismutase of Bacillus stearothermophilus, and were compared to activity measurements, in order to obtain some information on the structure and dynamics at Mn(III) present in the active site of the enzyme. The experimental data lead us to hypothesize the presence of two binding sites in the coordination sphere of the enzyme bound Mn(III), which are accessible to water and anions and have different chemical and spectroscopic properties. NMR measurements carried out in the presence of competitive inhibitors and the pH dependence of both NMR relaxation rates suggest that F-, N-3 and OH- ions bind to one site, while a water molecule binds to the other one. The stability constant values of the complexes between these anions and the enzyme are reported. The influence of the anions on activity and the pH dependence of NMR parameters are discussed.

Age dependence of the level of the enzymes involved in the protection against active oxygen species in the rat brain.

Levels of Cu, Zn superoxide dismutase (CuSOD), Mn superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPx) were assessed in the rat brain cortex. The concentrations of Cu- and MnSOD were found to increase linearly with the logarithm of the age of the animal from 3 days before birth to 30 months, both in the whole cortex tissue and in its cytoplasmic fraction. Catalase and GPx levels showed different trends; in particular, GPx, which appears to play a key role in detoxification of hydrogen peroxide, after an initial fall increases steadily with age. The enhancement of the levels of SOD and GPx could be related to protection against an increased production of reactive oxygen species in the aging process.

Characterization of free and immobilized amine oxidases.

Bovine plasma amine oxidase was covalently bound to CH-Sepharose 4B by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The immobilized enzyme showed no significant change in specific activity when spermidine was the substrate, while the enzyme affinity toward benzylamine and propylamine increased significantly. Similarly, the pig kidney diamine oxidase physically adsorbed to Con A-Sepharose showed large changes in affinity toward substrates such as p-dimethylaminoethylbenzylamine with respect to the native enzyme. These changes are discussed in terms of active site modification as a consequence of the enzyme immobilization.

Purification and characterization of amine oxidase from soybean seedlings.

A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being < or = 2%. The M(r) estimated by gel filtration is 113,000 and 77,000 by sodium lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.

Curie-point pyrolysis-gas chromatography/mass spectrometry in the art field. 2--The characterization of proteinaceous binders.

Curie point pyrolysis with gas chromatography/mass spectrometry has been employed to characterize some proteinaceous media used in the art field as painting binders: milk casein, egg yolk, egg albumin, bone glue, skin glue, rabbit glue and fish glue. A careful analysis of the gas chromatograms so obtained has led to the distinction of the different proteinaceous binders in terms of different chromatographic profiles. Some of the pyrolysis products have been identified by library search.

Succinylated copper, zinc superoxide dismutase. A novel approach to the problem of active subunits.

Bovine erythrocyte superoxide dismutase (BESOD) has been extensively succinylated with succinic anhydride. Succinylated BESOD has an identical electron paramagnetic resonance (EPR) spectrum but only 10% as much activity as the native enzyme, showing that an increase of the negative charge of the protein surface lowers the activity without alteration of the active site structure. On the other hand, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis indicates that interaction between subunits is strongly weakened by succinylation. NaDodSO4 has no effect on either the activity or EPR spectrum of the protein. BESOD was immobilized by coupling to a Sepharose matrix with no alteration of the EPR spectrum. Succinylation of the immobilized protein led to detachment from the gel of approximately 50% of the molecules, as estimated by parallel EPR measurements of the gel and activity determinations on the eluate. It is concluded the succinylation leads to dissociation of BESOD into nondenatured subunits, having lower activity than the native protein possibly because of charge effects on the enzyme-O2-interaction.

Kinetic characterization of soybean seedling amine oxidase.

The kinetic characterization of soybean seedling amine oxidase (SSAO) and the effect of ionic strength and pH on the enzyme activity have been studied. A strong dependence of the activity on the carbon chain length of alpha-omega diamines, peaking at C8 and at C5, for Km and kc, respectively, was found. The analysis of ionic strength effects on activity showed a high sensitivity of Km and an insensitivity of kc to this parameter. This behavior and the different dependence of Km and kc on the carbon chain length suggest a two-stage equilibrium model for the oxidative deamination of polyamines by SSAO. The formation of the first intermediate (complex amine-enzyme) is controlled mainly by ionic interactions and by the structure of the polyamine, while the formation of the second intermediate depends only on the length of the carbon chain of the diamine. The molecular dissociation constants of the system soybean seedling amine oxidase-cadaverine were obtained from the dependence of kc, Km, and kc/Km on pH. A mechanism of the initial steps of reaction, involving a two-stage equilibrium model, is proposed.

Effect of phosphate ion on the activity of bovine plasma amine oxidase.

The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 +/- 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 +/- 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.

Electrostatic control of oxidative deamination catalysed by bovine serum amine oxidase.

The ionic-strength-dependence of steady-state kinetic parameters (kc and Km') for non-biogenic (benzylamine, butylamine) and biogenic (spermine, spermidine) amines has been measured in the bovine serum amine oxidase reaction. The catalytic rate constant (kc) values are similar (0.9-2.5 s-1) for all the substrates studied and are almost constant over the experimental ionic strength range (24-155 mM). In contrast, Km' values are in the range 6-2300 microM and undergo a 4-12-fold increase with increasing ionic strength, parallelled by a decrease in catalytic efficiency. From an analysis of the kc and Km' values and their dependence on ionic strength, we conclude that more than one negative site is involved in the binding of these amines and that the relative dielectric constant of the binding site is lower than that of aqueous solutions.