The effects of buffer cations on interactions between mammalian copper-containing amine oxidases and their substrates.

We and others have observed that substrates for copper-containing amine oxidases cause substrate inhibition at high concentrations. Through use of a novel "pseudoquantitative" rapid equilibrium approach, kinetic analyses with human and bovine enzymes indicate that these effects are consistent with substrates binding to oxidised and reduced enzyme forms. Small cations compete with binding of substrates to oxidised and reduced enzyme, influencing both substrate turnover and substrate inhibition patterns. Cations reduce affinity of the resting bovine enzyme for spermidine, but not benzylamine, indicating that the predominant effect of cations on substrate oxidation results from binding to an anionic site outside the active site. However, binding of cations to the active site of the reduced form of both enzymes attenuates substrate inhibition with both spermidine and benzylamine. Our observations have significant practical implications for researchers assaying kinetic behaviour of these enzymes, and particularly those developing novel inhibitors of human copper-containing amine oxidases.

High resolution mini-gammacamera and 99mTc [HMPAO] - leukocytes for diagnosis of infection and radioguided surgery in diabetic foot.

Discovery of osteitis may be delayed because of late appearance of X-ray signs in patients with diabetic foot. Scintigraphy with labelled leukocytes is able to detect flogosis but often misses bone involvement, due to inadequate resolution of Anger camera, the commonest detector used in nuclear medicine. Radioguided surgery and biopsy with high resolution scintigraphy (HRS) started to be studied since 2000: although this method had never been tested for planning and guiding diabetic foot surgery, in our opinion it can help early diagnosis and surgical treatment of diabetic foot. Five patients with diabetic foot and suspected infection were studied with standard 99mTc [HMPAO]-leukocyte scan. In the same patients 2 mm spatial resolution HRS was performed 24 hours after administration of labelled WBC, using our inch2 field-of-view portable mini-gammacamera. Operations were done just after the 24h scan and were guided with the portable high resolution device in the four patients who showed positive scan. Scintigraphy with Anger camera and HRS were positive in four patients. HRS showed a bar-shaped radioactivity corresponding to small phalanges, close to the main inter-digital hot spot. The presence of osteitis on phalanges that had been shown by HRS was confirmed at surgery, that was successfully driven with the high resolution mini-camera. In conclusion HRS is able to diagnose early osteitis of diabetic foot and to guide diabetic foot surgery.

Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites.

Preparation, morphological characterization, and activity of thin films of horseradish peroxidase.

Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/SiO(2) or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60-90 A. By using a fluorescent label, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/SiO(2), corresponding to an estimated area per molecule of 2800 A(2) which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x10(12) molecules cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K(M) being of the order of 3-5x10(5) M(-1)s(-1) that is 1/20th of free HRP. The half-life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6 degrees C.

Enzyme mimics complexing Cu(II) ion: structure-function relationships.

Five peptides containing (His-X2)-His or (His-X3)-His motifs have been designed and synthesized to coordinate Cu(II). Structural information was obtained by various spectroscopic techniques and was used as constraint to search for local conformational energy minima by molecular mechanics. Thermodynamic stability constants of the Cu(II) chelates was obtained by 19F-NMR. The synthesized Cu(II)-peptide chelates were tested as catalysts of some important red-ox processes occuring in biological systems, in particular oxidation of ascorbate and dismutation of superoxide ion. The catalytic efficiency of the five chelates was much lower than that of ascorbate oxidase. On the contrary, two of them showed kinetic constants for superoxide dismutation about one order of magnitude lower than that of the enzyme Cu,Zn superoxide dismutase. In both cases, the catalytic properties were dependent on the peptide sequence. The relationships between structure and activity are discussed to find the structural parameters crucial for catalytic activity that can be modulated by appropriate design and synthesis of the peptides.

Purification and characterization of amine oxidase from pea seedlings.

A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.

Continuous flow immunosensor for atrazine detection.

The hapten atrazine was detected under continuous flow conditions using a micro-column which contained immobilized monoclonal antibodies (Ab) against atrazine and atrazine labeled with alkaline phosphatase (An*). The equilibrium of the antibody-hapten system, was achieved by a continuous flow of the tracer An* through the micro-column containing the immobilized antibodies. The activity of the tracer was monitored continuously, after the micro-column, by an amperometric detector using p-hydroquinone phosphate as substrate. When pulses of unlabeled atrazine (An) were added to the An* flowing continuously through the micro-column, An* bound to the antibody was displaced, with a consequent change of the detector signal. By this method atrazine concentrations in the range 9-180 micrograms/l were monitored under conditions of continuous operation. Since the equilibrium condition for the system Ab-An* was continuously restored by the flow of An* through the micro-column the regeneration of the antibody was not required.

Characterization of thin layers of glucose oxidase.

Glucose Oxidase (GOD) has been covalently bound to functionalized glass cover slips. The surface density of immobilized GOD molecules was measured by a method based on the amperometric determination of Flavin Adenine Dinucleotide (FAD). Atomic Force Microscopy (AFM) images, obtained in aqueous solution for the covalently bound enzyme, show a monomolecular layer of the enzyme on a functionalized glass surface. The catalytic constants were measured for the immobilized GOD and compared with those of the free enzyme.

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 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.

A sensitive spectrophotometry-based method for the determination of the rate of hydrogen peroxide generation in biological systems.

A new sensitive spectrophotometric method for the determination of the rate of hydrogen peroxide generation in biological systems has been developed. This method is based on the measurement of the oxidation rate of reduced cytochrome c by H2O2 in the presence of a mediator and permits the detection of H2O2 generation rates as low as 60 nM min-1. The solution of the differential equations of the kinetic process permitted the calculation of the kinetic rate constants and assessment of the conditions required to measure the hydrogen peroxide generation rate.

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.

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.

Isolation of amine oxidase from bovine plasma by a two-step procedure.

A novel method for isolation of amine oxidase from bovine plasma is reported; it involves a two-step procedure, namely ammonium sulfate fractionation and affinity chromatography with elution by aniline, which is a competitive inhibitor of the enzyme. A homogeneous enzyme, characterized by a specific activity of 0.44 U/mg, was obtained with a yield higher than 50%.

Immunological studies on chlorophyll-a/b proteins and their distribution in thylakoid membrane domains.

The immunological relationships between chlorophyll-a/b proteins from higher-plant thylakoid membranes have been studied by assaying purified chlorophyll proteins (CPs) with polyclonal and monoclonal antibodies. Although low levels of cross-reactions were observed between all light-harvesting proteins, the peripheral antennae (LHCII) were largely distinct from the inner antennae (CP 26 and CP 29). Chlorophyll-protein 24 and LHCI-680 have been proposed to have a role in connecting the inner and outer antennae, respectively, in photosystems I and II, and were closely related. The immunological relationships closely corresponded to the spectral properties. Antibodies were also used for locating chlorophyll-a/b proteins in grana, stroma and bundle-sheath membranes showing a strong lateral heterogeneity, which was maintained following State I State II transition. The only exception to this pattern was a specific LHCII population enriched in State-II stroma membranes. Chlorophyll proteins from bundlesheath chloroplasts, that have only cyclic electron flow, had epitopes distinct from those of their mesophyll homologues.