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ABSTRACT: Mitochondria are the dominant source of the cellular energy requirements through oxidative phosphorylation, but they are also central players in apoptosis. Nutrient availability may have been the main evolutionary driving force behind these opposite mitochondrial functions: production of energy to sustain life and release of apoptotic proteins to trigger cell death. Here, we explore the link between nutrients, mitochondria and apoptosis with known and potential implications for age-related decline and metabolic syndromes.
Aging 11/2012; · 5.13 Impact Factor
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ABSTRACT: A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X-100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30-35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M(w) /M(n) of 2.9. (1) H NMR, (13) C NMR, and FTIR analysis revealed the characteristic of the cis-1,4-polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition.
Biopolymers 08/2012; 97(8):589-94. · 2.87 Impact Factor
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ABSTRACT: The aminoaldehydes 4-aminobutanal and 5-aminopentanal, derived from the oxidation of the diamines putrescine and cadaverine,
and 1-(3-aminopropyl)-4-aminobutanal and aminodialdehyde, derived from the oxidation of the polyamines spermidine and spermine,
were produced utilizing a copper amine oxidase (CAO) from Euphorbia characias latex and tested with in vitro cultivation of Leishmaniainfantum promastigotes. Whereas the aminoaldehydes derived from the oxidation of the diamines were stimulating factors for growth
of Leishmaniainfantum promastigotes, the aldehydes derived from polyamines oxidation had a drastic inhibitory effect on the vitality and growth
of these parasites. Thus, a double scenario arises, showing the use of aldehydes from diamines to obtain a large number of
organisms of Leishmaniainfantum promastigotes to use in serological studies, whereas the aldehydes derived from polyamines could be used as a new strategy
for therapeutic treatment against these parasites.
Medicinal Chemistry Research 04/2012; 19(1):77-83. · 1.27 Impact Factor
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ABSTRACT: This minireview deals of a protein, a class III secreted peroxidase, present as unique isoform in the latex of the perennial Mediterranean shrub Euphorbia characias. The paper reports on the molecular properties, on the structures (primary, secondary and tertiary), and on the catalytic mechanism of this enzyme. Here is also reported the extraordinary effect of calcium ions on the structure and on the enzyme activity of Euphorbia peroxidase. These ions can either enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to execute different metabolic pathways toward the same substrate. This review will give a valuable reference to the peroxidase fans and the general readers will find many thorough suggestions for future researches giving birth to new studies and important discoveries.
The Protein Journal 02/2011; 30(2):115-23. · 1.04 Impact Factor
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ABSTRACT: This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources.
Biochemistry research international. 01/2011; 2011:369484.
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ABSTRACT: The oxidation of the pseudohalide thiocyanate (SCN(-)) by Euphorbia peroxidase, in the presence or absence of added calcium, is investigated. After incubation of the native enzyme with hydrogen peroxide, the formation of Compound I occurs and serves to catalyze the thiocyanate oxidation pathways. The addition of a stoichiometric amount of SCN(-) to Compound I leads to the native enzyme spectrum; this process clearly occurs via two electron transfers from pseudohalide to Compound I. In the presence of 10 mM calcium ions, the addition of a stoichiometric amount of SCN(-) to Compound I leads to the formation of Compound II that returns to the native enzyme after addition of a successive stoichiometric amount of SCN(-), indicating that the oxidation occurs via two consecutive one-electron transfer steps. Moreover, different reaction products can be detected when the enzyme-hydrogen peroxide-thiocyanate reaction is performed in the absence or presence of 10 mM Ca(2+) ions. The formation of hypothiocyanous acid is easy demonstrated in the absence of added calcium, whereas in the presence of this ion, CN(-) is formed as a reaction product that leads to the formation of an inactive species identified as the peroxidase-CN(-) complex. Thus, although monomeric, Euphorbia peroxidase is an allosteric enzyme, finely tuned by Ca(2+) ions. These ions either can enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to exploit different metabolic pathways toward the same substrate.
Biochemistry 10/2010; 49(40):8739-47. · 3.42 Impact Factor
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ABSTRACT: Tyramine, an important plant intermediate, was found to be a substrate for two proteins, a copper amine oxidase and a peroxidase from Euphorbia characias latex. The oxidation of tyramine took place by two different mechanisms: oxidative deamination to p-hydroxyphenylacetaldehyde by the amine oxidase and formation of di-tyramine by the peroxidase. The di-tyramine was further oxidized at the two amino groups by the amino oxidase, whereas p-hydroxyphenylacetaldehyde was transformed to di-p-hydroxyphenylacetaldehyde by the peroxidase. Data obtained in this study indicate a new interesting scenario in the metabolism of tyramine.
Archives of Biochemistry and Biophysics 08/2008; 475(1):18-24. · 2.93 Impact Factor
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ABSTRACT: A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron-protoporphyrin IX pentacoordinated with a histidine 'proximal' ligand as heme prosthetic group. In addition, the purified peroxidase contained 1 mole of endogenous Ca(2+) per mole of enzyme, and in the presence of excess Ca(2+), the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni(2+) causes reversible inhibition, whereas, in the presence of excess Ca(2+), Ni(2+) leads to an increase of the catalytic activity of Euphorbia peroxidase. UV/visible absorption spectra show that the heme iron remains in a quantum mechanically mixed-spin state as in the native enzyme after addition of Ni(2+), and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni(2+). In the presence of H(2)O(2) and in the absence of a reducing agent, Ni(2+) decreases the catalase-like activity of Euphorbia peroxidase and accelerates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni(2+) affects the H(2)O(2)-binding site and inhibits the formation of compound I. In the presence of excess Ca(2+), Ni(2+) accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni(2+)-binding sites with opposite functional effects.
FEBS Journal 04/2008; 275(6):1201-12. · 3.79 Impact Factor
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ABSTRACT: The interaction of xenon with copper/6-hydroxydopa (2,4,5-trihydroxyphenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediterranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. (129)Xe chemical shift provided evidence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the analyzed plant amine oxidases showed that the k(c) value of the xenon-treated enzymes was reduced by 40%. Moreover, whereas the measured K(m) value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the K(m) value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the k(c) value of the xenon-treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramatically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity.
FEBS Journal 06/2007; 274(10):2585-95. · 3.79 Impact Factor
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ABSTRACT: A new procedure to purify to homogeneity an Euphorbia characias latex peroxidase is performed employing a calmodulin-Sepharose column as affinity chromatography. The advantage of this approach is the isolation of a peroxidase under fast and mild elution conditions with a high recovery in total activity.
The Italian journal of biochemistry 04/2007; 56(1):1-5.
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ABSTRACT: Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN(-) in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN(-) treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. Ni(II) and Zn(II) do not bind the apo-protein in contrast to Co(II) which is incorporated to the same extent as Cu(II). However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.
FEBS Letters 09/2006; 580(18):4317-24. · 3.54 Impact Factor
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ABSTRACT: The reaction of Euphorbia characias latex peroxidase (ELP) with hydrogen peroxide as the sole substrate was studied by conventional and stopped-flow spectrophotometry. The reaction mechanism occurs via three distinct pathways. In the first (pathway I), ELP shows catalase-like activity: H2O2 oxidizes the native enzyme to compound I and subsequently acts as a reducing substrate, again converting compound I to the resting ferric enzyme. In the presence of an excess of hydrogen peroxide, compound I is still formed and further reacts in two other pathways. In pathway II, compound I initiates a series of cyclic reactions leading to the formation of compound II and compound III, and then returns to the native resting state. In pathway III, the enzyme is inactivated and compound I is converted into a bleached inactive species; this reaction proceeds faster in samples illuminated with bright white light, demonstrating that at least one of the intermediates is photosensitive. Calcium ions decrease the rate of pathway I and accelerate the rate of pathways II and III. Moreover, in the presence of calcium the inactive stable verdohemochrome P670 species accumulates. Thus, Ca2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.
Biological Chemistry 06/2006; 387(5):559-67. · 2.96 Impact Factor
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Francesca Pintus
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ABSTRACT: Peroxidases (EC 1.11.1.1-16) are a group of oxidoreductases utilizing hydrogen peroxide, or other peroxides, to oxidize a second reducing substrate belonging to a large array of organic and inorganic compounds. A class III peroxidase isolated from the latex of Mediterranean shrub Euphorbia characias (ELP), has peculiar features with respect to the other eme-peroxidases. In fact ELP is the first example of peroxidase that responds as an on/off switch to variation in the external Ca2+ level and represents the first peroxidase regulated by the Ca2+/calmodulin system.
The catalytic mechanism of ELP was well studied and characterized. In the present thesis we investigate the catalyc pathways of ELP with hydrogen peroxide as the unique substrate and in both the presence and absence of calcium ions showing the presence oh three different pathways. Pathway I: ELP shows catalise-like activity catalyzing decomposition of hydrogen peroxide to water and oxygen; Pathway II involves the return to native enzyme with the concomitant formation of superoxyde anion; Pathway III involves an irresistible loss of enzyme activity, with hydrogen peroxide acting as a suicide substrate.
Because the importance of the Ca2+ ions to influence the catalytic activity of Euphorbia peroxidase, we investigate the effects of another divalent cation (Ni2+) on the catalytic pathways of native ELP and in the presence of calcium ions. The enzyme is strongly inhibited by nickel ions an the absence of Ca2+, whereas, in the presence of both Ca2+ and Ni2+, the enzymatic activity is enhanced. The reported results are compatible with the hypothesis that ELP has Ni2+-binding sites eith opposite functional effects.
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ABSTRACT: An authentic soluble metallo-protein nucleotide pyrophosphatase/phosphodiesterase (ELNPP) was purified to homogeneity from Euphorbia characias latex. The native protein had a molecular mass of 80 ± 5 kDa and was shown to be formed by two apparently identical subunits, each containing 1 Ca2+ and 1 Mg2+ ion. Whereas Mg2+ was shown to be strongly bound to the enzyme, Ca2+ was easily removed by treatment with EDTA. Ca2+-demetalated enzyme was shown to be almost totally inactive and the activity was fully restored incubating the demetalated ELNPP with Ca2+ ions. ELNPP exhibited hydrolytic activities toward pyrophosphate/phosphodiester bonds of a broad range of substrates and very efficiently hydrolyzed the artificial substrate thymidine 5′-monophosphate 4-nitrophenyl ester generating 4-nitrophenolate as a final product, and it has been used for enzyme kinetic experiments. ELNPP represents the first example of a nucleotide pyrophosphatase/phosphodiesterase enzyme purified from the latex of a plant belonging to the large genus Euphorbia.
Plant Science.
