Maria Luisa Di Paolo

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.

Interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool in very old age.

Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative burst and phagocytosis with subsequent less capability to destroy bacteria. In successful ageing (nonagenarians), neutrophil efficiency (phagocytosis) increases. After ingested microbes, aged neutrophils are less prone to undergo apoptosis favouring chronic inflammation. Moreover, the superoxide dismutase (SOD) activity, which is necessary in avoiding ROS produced by oxidative burst, is limited in ageing. The mechanisms of age-related changes in neutrophil function are not fully understood, taking also into account that nonagenarians escape infections in comparison with elderly. Zinc pool may be involved because it is pivotal for neutrophil efficiency and SOD activity. Since zinc also controls the inflammation, via IL-6 and soluble factor of gp130 (sgp130), we have assessed the possible interrelationship among oxidative burst, apoptosis, inflammation, SOD, adhesion molecule Mac-1 and zinc pool in elderly and in nonagenarians. The oxidative burst and the capacity to increase Mac-1 after PMA stimulation decrease both in elderly and nonagenarians, but the latter display a slight increased neutrophil induced apoptosis, decreased sgp130, increased SOD, and more neutrophil zinc content, as it occurs in young-adults. Significant correlation exists between sgp130 and zinc pool in very old age. These findings suggest lower chronic inflammation in nonagenarians, via more zinc available, with subsequent long-life survival. Therefore, a more correct interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool exists in successful ageing with subsequent more effectiveness to control the inflammatory response to pathogens.

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/SiO2 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 Å. By using a fluorescent label, we calculated a protein density of 3.6 × 1012 molecules cm−2 on Si/SiO2, corresponding to an estimated area per molecule of 2800 Å2 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 ×1012 molecules cm−2). The enzyme immobilized on both supports showed a kcat/KM being of the order of 3–5×105 M−1s−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°C.

Electrostatic compared with hydrophobic interactions between bovine serum amine oxidase and its substrates.

A steady-state kinetic study of bovine serum amine oxidase activity was performed, over a wide range of pH values (5.4-10.2) and ionic strength (10-200 mM), using various (physiological and analogue) substrates as specific probes of the active-site binding region. Relatively small changes in k (cat) values (approx. one order of magnitude) accompanied by marked changes in K(m) and k(cat)/K(m) values (approx. six orders of magnitude) were observed. This behaviour was correlated with the presence of positively charged groups or apolar chains in the substrates. In particular, it was found that the docking of the physiological polyamines, i.e. spermidine and spermine, appears to be modulated by three amino acid residues of the active site, which we have named L(-)H(+), G(-)H(+) and IH(+), characterized by p K (a) values of 6.2+/-0.2 [Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231-2239], 8.2+/-0.3 and 7.8+/-0.4 respectively. The electrostatic interaction between the protonated substrates and the enzyme containing the residues L(-)H(+), G(-)H(+) and IH(+) in the deprotonated form, the on/off role of the IH(+) residue and the role of hydrophobic interactions with substrates characterized by apolar chains are discussed.

A senstive spectrophotometry-based mehtod 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.

Amino oxidase amperometric biosensor for polyamines

Abstract An improved amino oxidase enzyme electrode has been constructed and applied to the determination of the amount of polyamines present in real samples. The electrode is based on the amperometric detection of H 2 O 2 produced in the enzymatic oxidation of polyamines by amino oxidase. Amino oxidase from soybean seedlings, characterized by an extremely high activity for cadaverine and putrescine, was used. The enzyme was immobilized in an agarose matrix in the presence of glutaraldehyde and bovine serum albumin on the surface of a Pt electrode. Cadaverine, in concentrations between 0.5 and 500 μM, can be quantitatively determined by use of the amino oxidase electrode, the linear calibration range being 0.5–10 μM. The lower detection limit was 0.2 μM and the response time was 15 to 60 s. Putrescine showed similar behaviour. The maximum current response for cadaverine was 5.1 μA/cm 2 , with an apparent Michaelis—Menten constant ( K m ′) of 0.175 mM. The sensor response was stable for more than 32 hours of continuous operation at room temperature and, in the presence of fish or meat homogenates, no change in the signal-to-noise ratio was observed. The long-term stability, pH and temperature response of the biosensor has also been studied.

TAT-Mediated Aequorin Transduction: An Alternative Approach for Effective Calcium Measurements in Plant Cells

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.

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%.

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.

Crystallization and preliminary X-ray data of amine oxidase from bovine serum

A copper-containing amine oxidase extracted from bovine serum (BSAO) and purified to homogeneity has been deglycosylated and crystallized. The crystals obtained belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 77.68, b = 131.19, c = 134.00 A, and diffract to at least 2.4 A resolution. BSAO is the first mammalian amine oxidase to be crystallized.