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In 1990s, the European spiny lobster

Overweight and obesity are constantly increasing, not only in Western countries but also in low-middle-income ones. The decrease of both the intake of carbohydrates and their assimilation are among the main dietary strategies to counter these conditions. α-Amylase, a key enzyme involved in the digestion of carbohydrates, is the target enzyme to reduce the absorption rate of carbohydrates. α-Amylase inhibitors (α-AIs) can be found in plants. The common bean, Phaseolus vulgaris is of particular interest due to the presence of protein-based α-AIs which, through a protein-protein interaction, reduce the activity of this enzyme. Here we describe the nature of the various types of common bean seed extracts, the type of protein inhibitors they contain, reviewing the recent Literature about their molecular structure and mechanism of action. We also explore the existing evidence (clinical trials conducted on both animals and humans) supporting the potential benefits of this protein inhibitors from P. vulgaris, also highlighting the urgent need of further studies to confirm the clinical efficacy of the commercial products. This work could contribute to summarize the knowledge and application of P. vulgaris extract as a nutraceutical strategy for controlling unwanted weight gains, also highlighting the current limitations.

Vitellogenin is an essential protein involved in ovary maturation in many animals. Detection of this protein correlated with reproductive capacity may be important if carried out on marine organisms such as the red spiny lobster Palinurus elephas, a crustacean economically important crop from wild fish catches. Moreover, in recent years, vitellogenin has assumed an important role as a possible biomarker of marine environmental pollution, as its expression levels can be influenced by the presence of similar oestrogen pollutants and affect the reproductive sphere of marine organisms such as crustaceans. The P. elephas vitellogenin protein and its coding gene have never been isolated, so there is little information about its presence in this lobster. The aim of the present study was to develop a molecular strategy to create, for the first time, an antibody for the detection and quantization of vitellogenin in P. elephas.

In 1990s, the European spiny lobster Palinurus elephas, one of the most commercially important species in the Mediterranean, exhibited a population decline. For this reason, fully protected areas (FPAs) appeared effective in re-establishing natural populations and supporting fishery-management objectives. Here, the reproductive parameters of P. elephas populations in two different FPAs (Su Pallosu and Buggerru, central-western Mediterranean), where a restocking programme was carried out, and in their surrounding commercial zones, were investigated from quantitative and qualitative perspectives. The comparison of fecundity between females collected inside and outside FPAs did not show statistical differences as well as the vitellogenin concentration, which did not vary among eggs of different size classes of females caught inside and outside the FPAs, indicating the same reproductive potential. The study demonstrated a benefit of overexploited populations in terms of enhancement of egg production overtime (15 years for Su Pallosu and 6 years for Buggerru) with a mean egg production 4.25–5.5 times higher at the end of the study than that observed at the beginning of the study. The main driver of eggs production appeared to be size, with larger lobsters more present inside the FPAs than outside. Given these results, the dominant contribution of the two studied FPAs to the regional lobster reproduction is remarkable.

Polyphenol oxidase (PPO, E.C. 1.14.18.1) is a nearly ubiquitous enzyme that is widely distributed among organisms. Despite its widespread distribution, the role of PPO in plants has not been thoroughly elucidated. In this study, we report for the absence of PPO in Cynomorium coccineum, a holoparasitic plant adapted to withstand unfavorable climatic conditions, growing in Mediterranean countries and amply used in traditional medicine. The lack of PPO has been demonstrated by the absence of enzymatic activity with various substrates, by the lack of immunohistochemical detection of the enzyme, and by the absence of the PPO gene and, consequently, its expression. The results obtained in our work allow us to exclude the presence of the PPO activity (both latent and mature forms of the enzyme), as well as of one or more genes coding for PPO in C. coccineum. Finally, we discuss the possible significance of PPO deficiency in parasitic plants adapted to abiotic stress.

This study is the first molecular and biochemical analysis conducted on Pompia, a plant of unknown origin that is endemic to Sardinia; this plant is thought to belong to the Citrus genus. Here, genes coding for the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), and polyphenol oxidase (PPO, EC 1.14.18.1) were identified. We detected the aforementioned enzymes in fresh leaf tissue and assessed the catalytic activity of each to support the molecular and biochemical data. This was the first molecular study to define the primary structure of proteins with antioxidant activity in Pompia. The study also contributed to the enrichment of gene databases and created the basis for molecular phylogenetic studies, which is important because this plant currently has no taxonomic or phylogenetic classification.

Polyphenol oxidase (PPO, E.C. 1.14.18.1) is a nearly ubiquitous enzyme that is widely distributed among organisms. Despite its widespread distribution, the role of PPO in plants has not been thoroughly elucidated. In this study, we report for the absence of PPO in Cynomorium coccineum, a holoparasitic plant adapted to withstand unfavorable climatic conditions, growing in Mediterranean countries and amply used in traditional medicine. The lack of PPO has been demonstrated by the absence of enzymatic activity with various substrates, by the lack of immunohistochemical detection of the enzyme, and by the absence of the PPO gene and, consequently, its expression. The results obtained in our work allow us to exclude the presence of the PPO activity (both latent and mature forms of the enzyme), as well as of one or more genes coding for PPO in C. coccineum. Finally, we discuss the possible significance of PPO deficiency in parasitic plants adapted to abiotic stress.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. SS new components of the family were characterized by top-down liquid chromatography mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S-1 -> A, P-Ko P-36 -> S, and P-Ko A(41) -> S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

The N-glycosylation in pea seedling amine oxidase and lentil seedling amine oxidase was analyzed in the present work. For that purpose, the enzymes were purified as native proteins from their natural sources. An enzymatic deglycosylation of pea seedling amine oxidase by endoglycosidase H under denaturing conditions combined with its proteolytic digestion by trypsin was carried out in order to analyze both N-glycans and "trimmed" N-glycopeptides with a residual N-acetylglucosamine attached at the originally occupied N-glycosylation sites. The released N-glycans were subjected to a manual chromatographic purification followed by MALDI-TOF/TOF MS. MS and MS/MS analyses were also performed directly on peptides and N-glycopeptides generated by proteolytic digestion of the studied enzymes. Sequencing of glycopeptides by MALDI-TOF/TOF MS/MS after their separation on a RP using a microgradient chromatographic device clearly demonstrated binding of paucimannose and hybrid N-glycan structures at Asn558. Such carbohydrates have been reported to exist in many plant N-glycoproteins, e.g. in peroxidases. Although high-mannose glycan structures were identified after the enzymatic deglycosylation, they could not be assigned to a particular N-glycosylation site. The presence of unoccupied glycosylation sites in several peptides was also confirmed from MS/MS results.

A copper- and zinc-containing superoxide dismutase (Cu/Zn-SOD) cDNA was isolated from the Euphorbia characias latex (Elx) using consensus degenerate hybrid oligonucleotide primer (CODEHOP) design and RT-PCR strategy. Both 3'and 5'untraslated regions (UTR) were isolated by rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the Cu/Zn-SOD cDNA contains an open reading frame encoding a protein of 152 amino acids. Bioinformatic analyses of Elx SOD gene revealed a high identity rate with a large number of plant Cu/Zn-SODs in the deduced amino acid sequence. Since isoenzymes may be generated through the multiplicity of SOD genes or result from post-trascriptional events, genomic Southern blot in conjunction with northern blot experiments were also performed. The genomic analysis showed that the E. characias genome contains a single SOD gene. Northern blot analyses confirmed the presence of a single SOD mRNA demonstrating that alternative splicing does not occur. Quantita- tive real time-PCR (qRT-PCR) experiments showed that SOD gene expression in latex reaches maximum levels during the summer. These results suggest that the Cu/Zn-SOD of Euphorbia characias latex probably may be involved in the antioxid- ative process triggered by oxidative stress induced by the conditions of environmental change in which the plant lives.

The PROP taste phenotype varies greatly among individuals, influencing eating behavior and therefore may play a role in body composition. This variation is associated with polymorphisms in the bitter receptor gene TAS2R38 and the taste-bud trophic factor gustin gene. The aim of this study was to examine the relationship between TAS2R38 haplotypes and the gustin gene polymorphism rs2274333 in modulating PROP taste phenotype. PROP phenotype was determined in seventy-six volunteers (29 males, 47 females, age 25±3 y) by scaling methods and threshold measurements. TAS2R38 and gustin gene genotyping was performed using PCR techniques. The lowest responsiveness in PROP nontasters is strongly associated with the AVI nontasting TAS2R38 variant and the highest responsiveness in supertasters is strongly associated to allele A and genotype AA of the gustin gene. These data support the hypothesis that the greater sensitivity of supertasters could be mediated by a greater taste-bud density. Polymorphisms in TAS2R38 and gustin gene, together, accounted for up to 60% of the phenotypic variance in PROP bitterness and to 40% in threshold values. These data, suggest that other unidentified factors may be more relevant for detecting low concentrations of PROP. Moreover, the presence of the PAV variant receptor may be important for detecting high concentrations of PROP, whereas the presence of allele A in gustin polymorphism may be relevant for perceiving low concentrations. These data show how the combination of the TAS2R38 and gustin gene genotypes modulate PROP phenotype, providing an additional tool for the evaluation of human eating behavior and nutritional status.

o-Aminophenols have been long recognised as tyrosinase substrates. However their exact mode of interaction with the enzyme's active site is unclear. Properly vic-substituted o-aminophenols could help gain some insight into tyrosinase catalytic mechanism. Methods: Eight vic-substituted o-aminophenols belonging to two isomeric series were systematically evaluated as tyrosinase substrates and/or activators and/or inhibitors, by means of spectrophotometric techniques and HPLC-MS analysis. Some relevant kinetic parameters have also been obtained. Results: Four o-aminophenolic compounds derived from 3-hydroxyorthanilic acid (2-amino-3-hydroxybenzenesulfonic acid) and their four counterparts derived from the isomeric 2-hydroxymetanilic acid (3-amino-2- hydroxybenzenesulfonic acid) were synthesised and tested as putative substrates for mushroom tyrosinase. While the hydroxyorthanilic derivatives were quite inactive as both substrates and inhibitors, the hydroxymetanilic compounds on the contrary all acted as substrates for the enzyme, which oxidised them to the corresponding phenoxazinone derivatives. General significance: Based on the available structures of the active sites of tyrosinases, the different affinities of the fourmetanilic derivatives for the enzyme, and their oxidation rates,we propose a new hypothesis regarding the interaction between o-aminophenols and the active site of tyrosinase that is in agreement with the obtained experimental results.

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.

The changes in the heme environment and overall structure occurring during reversible thermal inactivation and in denaturant guanidinium of Euphorbia characias latex peroxidase (ELP) were investigated in the presence and absence of calcium ions. Native active enzyme had an absorption spectrum typical of a quantum-mixed spin ferric heme protein. After 40 min at 60 degrees C ELP was fully inactivated showing the spectroscopic behavior of a pure hexacoordinate low-spin protein. The addition of Ca2+ to the thermally inactivated enzyme restored its native activity and its spectroscopic features, but did not increase the stability of the protein in guanidinium. It is concluded that, in Euphorbia peroxidase, Ca2+ ion play a key role in conferring structural stability to the heme environment and in retaining active site geometry.

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca(2+), CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca(2+)/CaM-activated proteins are known to tune the interaction between calcium and H(2)O(2) in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) [Medda et al. (2003) Biochemistry 42, 8909-8918]. Here we report the cDNA nucleotide sequence of Euphorbia latex peroxidase, showing that the derived protein has two distinct amino acid sequences recognized as CaM-binding sites. The cDNA encoding for an E. characias CaM was also found and sequenced, and its protein product was detected in the latex. Results obtained from different CaM-binding assays and the determination of steady-state parameters showed unequivocally that ELP is a CaM-binding protein activated by the Ca(2+)/CaM system. To the best of our knowledge, this is the first example of a peroxidase regulated by this classic signal transduction mechanism. These findings suggest that peroxidase might be another node in the Ca(2+)/H(2)O(2)-mediated plant defense system, having both positive and negative effects in regulating H(2)O(2) homeostasis.

In this review the characteristics of the prosthetic group and the role of copper in amine oxidase purified from lentil seedlings are compared with the corresponding features of the amine oxidase isolated from bovine serum. Although both enzymes contain the same organic cofactor, the 6-hydroxydopa (2,4,5-trihydroxyphenethylamine) quinone, the catalytic cycle of lentil seedling amine oxidase operates through a Cu(I)-free-radical intermediate of the cofactor, whereas in bovine serum enzyme the radical form was not observed. The role of the metal in the catalytic mechanism of the two enzymes is also discussed. Moreover, the energetic domains and the effect of the temperature on activity, for both enzymes, are examined using differential scanning calorimetry.

A cDNA encoding for a copper containing amine oxidase has been isolated and sequenced from young leaves of Euphorbia characias, a perennial mediterranean shrub. A single long open reading frame of 2068 pb encodes a protein composed of 653 amino acids with a molecular mass of about 74 kDa. A putative 24-aminoacid signal peptide precedes the sequence of the mature protein, with characteristics of a secretion signal peptide. Alignments of Euphorbia amine oxidase cDNA nucleotide sequence with that of amine oxidase from the seedlings of the pulses lentil, pea, and chickpea reveal several conserved regions, especially in the C-terminus, with a homology 90%-97%. The near 5' region shows several insertions, deletions, and different nucleotide sequence with ca. 60% homology. The enzyme contains 1%-2% carbohydrate deduced by deglycosylation experiments. Five cysteine residues are present in the deduced aminoacid sequence with a single disulfide bridge as judged by titration with cysteine reagents.

Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.

Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).

This article reviews and comments on the physiological roles of ceruloplasmin (Cp). We show that, in addition to its ascertained involvement in iron homeostasis, the protein, by virtue of its unique structure among multicopper oxidases, is likely involved in other processes of both an enzymatic and a nonenzymatic nature. In particular, based on the analysis of the kinetic parameters, on the one hand, and of the side-products of the oxidation, on the other, we propose that the long-recognized ability of Cp to interact with and oxidize non-iron substrates may be of physiological relevance. The striking example of 6-hydroxydopamine oxidation is presented, where we show that the catalytic action is carried out readily under physiological conditions, without release of potentially toxic oxygen intermediates.

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.

A nucleotide pyrophosphatase (EC 3.6.1.9) was purified to homogeneity from lentil seedlings. The enzyme is a single polypeptide chain of 75 +/- 2 kDa that exhibits hydrolytic activities toward pyrophosphate linkages of several substrates. Reduced and oxidized forms of NAD(P) were shown to be hydrolyzed to nicotinamide mononucleotide and AMP. Other dinucleotides such as FAD and dinucleoside oligophosphates were hydrolyzed as well, but with lower efficiency. Pyrophosphatase activity was increased in the presence of divalent cations such as Ca2+, Mg2+, and Mn2+, whereas Cu2+, Zn2+, and Ni2+ ions inhibited this activity. The active site in the enzyme was not defined, but histidine residue(s) seemed to be crucial for the enzymatic activity.

Copper amine oxidase was shown to be able to catalyse the oxidative deamination of 2-, 3- and 4-Br-derivatives of benzylamine to the corresponding aldehydes, that all absorb at 250 nm. This change in the absorption spectrum made it possible to follow the enzyme reaction. 2-Br-benzylamine, 3-Br-benzylamine, and 4-Br-benzylamine showed Km values similar to benzylamine, but 3-Br-benzylamine showed a slower kc, which allows it to be a catalytically more efficient substrate. Under anaerobic conditions the native enzyme oxidised 1 equivalent of all Br-derivatives and released 1 equivalent of aldehyde per enzyme subunit. These findings demonstrate that, in anaerobic conditions, the enzyme can oxidise substrates with a single incomplete turnover. The possible involvement of the cofactor 6-hydroxydopa quinone and of a negatively charged residue in the oxidation of Br-benzylamines is discussed.

The oxidation of L-ornithine and L-arginine catalyzed by lentil (Lens esculenta) seedling copper-amine oxidase has been investigated by polarographic techniques, optical spectroscopy, and capillary electrophoresis. Both L-ornithine and L-arginine were found to be poor substrates for lentil amine oxidase. L-Ornithine was oxidized to glutamate-5-semialdehyde and ammonia, in similar manner as usual substrates. Glutamate-5-semialdehyde spontaneously cyclizes to delta1-pyrroline-5-carboxylic acid. Arginine is oxidized by an unusual mechanism yielding glutamate-5-semialdehyde, ammonia, and urea as reaction products.

The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k(c) value is different from that of native or Cu-fully-reconstituted enzyme, while K(m) is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 105m−1cm−1 and 6000 m−1cm−1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.

Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. γ-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.

Spermine is a substrate of lentil seedling amine oxidase and is oxidized at terminal amino groups to a dialdehyde: 2 mol of hydrogen peroxide and two mol of ammonia per mol of spermine are formed. In the presence of high amounts of spermine, the aldehydic groups formed upon oxidation of spermine by the enzyme, may react with primary amino groups of free spermine leading to the formation of aromatic pyrimidinic ring after beta-elimination at secondary amino groups.

Superoxide dismutase has been purified to homogeneity from Oryza sativa germinated seeds growth in the dark. The purified enzyme contained two electrophoretically distinct bands on continuous gel electrophoresis or analytical gel electrofocusing. SDS-PAGE showed a single band of an M(r) 15000 while gel chromatography on Sephadex G 100 showed a single peak of an M(r) 32000. It contained two Cu and two Zn ions. The spectra of ultraviolet and visible regions were similar to those of Cu-Zn mammalian and plant superoxide dismutases. The activation energy was estimated at 16 Kcal mol-1.

In this review, the widely distributed plant copper-amine oxidases are described. The purification procedures, molecular features, substrate specificities, inhibitors, the stoichiometry of the catalysed reaction, spectroscopic features, the prosthetic groups and reaction mechanisms, are all reviewed.

Peroxidase (E.C. 1.11.1.7., hydrogen donor oxidoreductase) is widely distributed and has been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance. However the role that it plays in metabolism is not clear due to the large number of reactions it catalyzes and the considerable number of isozymic species (2). In tomato plants, Evans and Aldridge (3) separated out six isoperoxidases and in a later paper Evans reported 12 isoperoxidases from tomato shoots (4). A homogeneous tomato fruit peroxidase isozyme was obtained by Jen et al. (5) using hydrophobic chromatography. Isozymes were not detected in Euphorbia characias peroxidase (6), in Ipomoea batatas peroxidase (7) and in Hordeum vulgare peroxidase (8). The simultaneous presence of Cu (II) amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (6,8,9). In the graminea Oryza sativa, widely distributed, an FAD amine oxidase is present that oxidizes diamines (10). In this plant we also found two isoperoxidases called perox I and II. Only perox I was purified to homogeneity and its enzymatic, physical and chemical properties have been studied.

The N-glycosylation in pea seedling amine oxidase and lentil seedling amine oxidase was analyzed in the present work. For that purpose, the enzymes were purified as native proteins from their natural sources. An enzymatic deglycosylation of pea seedling amine oxidase by endoglycosidase H under denaturing conditions combined with its proteolytic digestion by trypsin was carried out in order to analyze both N-glycans and "trimmed" N-glycopeptides with a residual N-acetylglucosamine attached at the originally occupied N-glycosylation sites. The released N-glycans were subjected to a manual chromatographic purification followed by MALDI-TOF/TOF MS. MS and MS/MS analyses were also performed directly on peptides and N-glycopeptides generated by proteolytic digestion of the studied enzymes. Sequencing of glycopeptides by MALDI-TOF/TOF MS/MS after their separation on a RP using a microgradient chromatographic device clearly demonstrated binding of paucimannose and hybrid N-glycan structures at Asn558. Such carbohydrates have been reported to exist in many plant N-glycoproteins, e.g. in peroxidases. Although high-mannose glycan structures were identified after the enzymatic deglycosylation, they could not be assigned to a particular N-glycosylation site. The presence of unoccupied glycosylation sites in several peptides was also confirmed from MS/MS results.

The rate of autoxidation of 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine) and the sensitivity of this autoxidation to increase by peroxidases was studied. The autoxidation of 6-hydroxydopamine proceeds by a free radical chain reaction involving O2 and produces the corresponding chromogen 6-hydroxydo-paminequinone and hydrogen peroxide. The yield of quinone produced increased with increasing pH in the range 5 → 9. The ability of peroxidases to increase the autoxidation of 6-hydroxydopamine at pH 5.5 has been used as sensitive assay for these enzymes. The results are tabulated and discussed.

The ability to perceive the bitter taste of 6-n-propylthiouracil (PROP) is a variable phenotype that has been associated with body mass index (in kg/m(2)) and linked to food choice and satiety. PROP-sensitive and -nonsensitive individuals are defined as tasters and nontasters, respectively. Sensitivity to PROP is a heritable trait based on the TAS2R38 gene on chromosome 7q34. In a recent study we demonstrated an association between PROP sensitivity and the single-nucleotide polymorphism (SNP) rs2274333 (+292A/G) within a coding sequence of the gustin/carbonic anhydrase VI gene. The purpose of this study was to develop a rapid and inexpensive screening method for identification of the rs2274333 SNP in individuals with varying sensitivity to PROP. Our results show that the methodology employed allows distinguishing A/G alleles perfectly, with a simple DNA digestion of a polymerase chain reaction fragment covering the SNP site of interest. So, the polymerase chain reaction followed by restriction fragment length polymorphism assay described in this article can be used as an alternative to sequencing in bitter taster status research, and could be employed as a survey tool in nutrigenomic studies.

Peroxidases (E.C. 1.11.1.7., hydrogen donor oxidoreductase) utilize hydrogen peroxide or substituted peroxides for the oxidation of a large number of substrates. Peroxidases are widely distributed and have been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance, but the physiological functions and metabolic control of these enzymes are still poorly understood. The simultaneous presence of amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (2,3). Recently we have purified an amine oxidase from Hordeum vulgare (4) and we have attempted to purify the peroxidase in order to study in vitro the reconstituted coupled system. beta-glucosidase (beta-D-glucoside glucohydrolase E.C. 3.2.1.21.) is capable of transforming glucosides in glucose and the corresponding aglycone or disaccharides as cellobiose, sophorose, gentiobiose. This enzyme is widely distributed in plants, fungi, bacteria, yeasts and animals (5,6). In the homogenate of Hordeum vulgare seedlings we also found beta-glucosidase activity and also attempted to purify beta-glucosidase. This enzyme copurified with peroxidase up to the last step. We report here the isolation of peroxidase and beta-glucosidase from Hordeum vulgare seedlings: some molecular and kinetic properties are given.

Here we report the cDNA nucleotide sequences of a calmodulin-binding catalase and an antiquitin from the latex of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent additional nodes in the Euphorbia defense systems, and a multi-enzymatic interaction contributing to plant’s protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers.

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.

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: H 2 O 2 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, Ca 2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.

Plant copper/quinone amine oxidases are homodimeric enzymes containing Cu II and a quinone derivative of a tyrosyl residue (2,4,5-trihydroxyphenylalanine, TPQ) as cofactors. These enzymes catalyze the oxidative deamination of primary amines by a classical ping–pong mechanism, i.e. two distinct half-reactions, enzyme reduction by substrate followed by its re-oxidation by molecular oxygen. In the first half-reaction two forms of the reduced TPQ have been observed, the colorless Cu II -aminoquinol and the yellow Cu I -semiquinolamine radical so that this enzyme may be referred to as a “protein-radical enzyme”. The interaction of xenon, in aqueous solutions, with the copper/TPQ amine oxidase from lentil ( Lens esculenta ) seedlings has been investigated by NMR and optical spectroscopy. NMR data indicate that xenon binds to the protein. Under 10 atm gaseous xenon and in the absence of substrates more than 60% native enzyme is converted into Cu I -semiquinolamine radical species, showing for the first time that both monomers in the dimer can generate the radical. Under the same experimental conditions the copper-free lentil enzyme is able to generate an intermediate absorbing at about 360 nm, which is assigned to the product Schiff base quinolaldimine which, to the best of our knowledge, has never been observed during the catalytic mechanism of plant amine oxidases. A possible role of the lysine residue responsible for the formation of Cu I -semiquinolamine and quinolaldimine, is proposed.

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2-enyl) adenine, N6-(4-amino-cis-but-2-enyl) adenine and N6-(4-aminobut-2-ynyl) adenine. From these, N6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (Km approximately 10(-4)M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (Ki approximately 10(-5) - 10(-4) M) whereas, N6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.

Copper/quinone amine oxidases contain Cu(II) and the quinone of 2,4,5-trihydroxyphenylalanine (topaquinone; TPQ) as cofactors. TPQ is derived by post-translational modification of a conserved tyrosine residue in the protein chain. Major advances have been made during the last decade toward understanding the structure/function relationships of the active site in Cu/TPQ amine oxidases using specific inhibitors. Mechanism-based inactivators are substrate analogues that bind to the active site of an enzyme being accepted and processed by the normal catalytic mechanism of the enzyme. During the reaction a covalent modification of the enzyme occurs leading to irreversible inactivation. In this review mechanism-based inactivators of plant Cu/TPQ amine oxidases from the pulses lentil (Lens esculenta), pea (Pisum sativum), grass pea (Lathyrus sativus) and sainfoin (Onobrychis viciifolia,) are described. Substrates forming, in aerobiotic and in anaerobiotic conditions, killer products that covalently bound to the quinone cofactor or to a specific amino acid residue of the target enzyme are all reviewed.

The catalysis of class III plant peroxidases is described based on the reaction scheme of horseradish peroxidase. The mechanism consists in four distinct steps: (a) binding of peroxide to the heme-Fe(III) to form a very unstable peroxide complex, Compound 0; (b) oxidation of the iron to generate Compound I, a ferryl species with a pi-cation radical in the porphyrin ring; (c) reduction of Compound I by one substrate molecule to produce a substrate radical and another ferryl species, Compound II; (d) reduction of Compound II by a second substrate molecute to release a second substrate radical and regenerate the native enzyme. Under unfavourable conditions some inactive enzyme species can be formed, known as dead-end species. Two calcium ions are normally found in plant peroxidases and appear to be important for the catalytic efficiency.

Peroxidase from Opuntia ficus indica fruits was purified with chromatographic methods. The enzyme had a characteristic spectrum in the visible region and R z ( A 403 A 275 ) value of 2.56. It showed a single band in SDS-PAGE electrophoresis. The peroxidase had a Mr of 58 000±2000, an isoelectric point of 7.2 and contained an ironprotoporphyrin IX as prosthetic group. The pH optimum was at 5.75 in 100 mM Na acetate buffer using o-dianisidine as substrate. The activation energy was estimated to be 16 kcal mol−1 and 50% inactivation occurred after 60 min at 60°.

Peroxidases are widely distributed and have been isolated from many higher-order plants, animal tissues, yeast and microorganisms. During measurements of peroxidase activities in samples of human plasma, we noticed the presence of a compound in the plasma which was interfering with the peroxidase assay. In this paper we describe the purification and characterization of this factor, which was identified as uric acid.The procedure used to purify uric acid from plasma involved ultra-filtration of the plasma, heat denaturation, DEAE-cellulose chromatography, and high performance liquid chromatography. The lyophilized powder was tested for homogeneity using an HPLC apparatus and capillary electrophoresis. Genuine uric acid samples were used for comparison.The compound obtained by the above-reported purification procedure was identified as uric acid by spectrophotometric analysis through comparison with genuine uric acid samples. Spectrophotometric measurements indicated that uric acid was degraded by HRP in the presence of H2O2.The experimental procedures described above allowed us to isolate and identify uric acid as the component in human plasma that acts as a true substrate for peroxidases.

Spermine is a substrate of lentil (Lens culinaris) seedling amine oxidase and the oxidation products are reversible inactivators of the enzyme. The spermine is oxidized at the terminal amino groups to a dialdehyde: 2 moles of hydrogen peroxide and 2 moles of ammonia per mole of spermine are formed. The pH optimum of the enzyme with spermine is 7.9 in TI-HCI buffer; the K(m) value is 4.4.10(-4) molar, similar to that found with other substrates (putrescine and spermidine).