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1.
Food Chem ; 297: 125005, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253325

RESUMO

Multiwalled carbon nanotubes molybdenum disulfide 3D nanocomposite (MWCNT-MoS2 NC) was successfully synthesized via eco-friendly hydrothermal method. The microstructural characterization of synthesized nanocomposite was carried out using different spectroscopic and microscopic techniques. Nanocomposite was activated using glutaraldehyde chemistry and used as a platform to immobilize Lens culinaris ß-galactosidase (Lsbgal) which resulted in 93% of immobilization efficiency. Attachment of Lsbgal onto nanocomposite was confirmed by AFM, FE-SEM, FTIR, and CLSM. The nanobiocatalyst showed broadening in operational pH and temperature working range. Remarkable increase in thermal stability was observed as compared to soluble enzyme. Nanobiocatalyst showed outstanding increase in storage stability, retained 92% of residual activity over a period of 8 months. This offers good reusability as it retained ∼50% residual activity up to 21 reuses and exhibited higher rate of lactose hydrolysis in whey. MWCNT-MoS2 NC conjugated to biomolecules can serve as a potential platform for fabrication of lactose biosensor.


Assuntos
Lactose/metabolismo , Lens (Planta)/enzimologia , Nanocompostos/química , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo , Biocatálise , Dissulfetos/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Molibdênio/química , Nanotubos de Carbono/química , Temperatura Ambiente , beta-Galactosidase/química
2.
Plant Physiol Biochem ; 108: 422-433, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27552180

RESUMO

Galactinol synthase (GS, EC 2.4.1.123) catalyzes the transfer of a galactosyl residue from UDP-galactose to myo-inositol to synthesize galactinol, a precursor for raffinose family oligosaccharides (RFO) biosynthesis. Screening, a cDNA library constructed with RNA isolated from developing lentil seeds, with partial GS genes resulted in identification of cDNA clones for two isoforms of GS, LcGolS1 (1336 bp, ORF-1002 bp, 334 amino acids) and LcGolS2 (1324bp, ORF-975bp, 325 amino acids) with predicted molecular weights of 38.7 kDa and 37.6 kDa, respectively. During lentil seed development, LcGolS1 transcripts showed higher accumulation during 26-32 days after flowering (DAF) corresponding to seed desiccation, while LcGolS2 showed maximum accumulation at 24 DAF, prior to increase in LcGolS1 transcripts. GS enzyme activity was maximum at 26 and 28 DAF and corresponded to galactinol accumulation, which also increased rapidly at 22 DAF with maximum accumulation at 26 DAF. Substrates for GS activity, myo-inositol and glucose/galactose were present in high concentrations during early stages of seed development but gradually decreased from 20 DAF to 32 DAF when galactinol concentration increased coinciding with increased GS enzyme activity.


Assuntos
Galactosiltransferases/metabolismo , Lens (Planta)/enzimologia , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Clonagem Molecular , DNA Complementar , Dissacarídeos/metabolismo , Galactosiltransferases/química , Galactosiltransferases/genética , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Lens (Planta)/genética , Lens (Planta)/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Conformação Proteica , Padrões de Referência , Reprodutibilidade dos Testes , Sementes/genética
3.
J Basic Microbiol ; 55(3): 346-53, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24740715

RESUMO

Soil bacteria are a new phytoremediation system for the removal of heavy metals from soils. In this study, fifteen soil bacteria were isolated from root nodules of lentil growing in heavy metals contaminated soils, particularly by lead. Molecular characterization of the collection showed a large diversity, including Agrobacterium tumefaciens, Rahnella aquatilis, Pseudomonas, and Rhizobium sp. These soil bacteria had a wide range of tolerance to heavy metals. Among them, strains of A. tumefaciens and R. aquatilis tolerated up to 3.35 mM Pb; whereas Pseudomonas tolerated up to 3.24 mM Pb. The inoculation of lentil grown hydroponically with inoculums formed by these efficient and Pb resistant bacteria enhanced plant biomass. The treatment of this symbiosis by 1 mM Pb for 10 days or by 2 mM Pb for 3 days demonstrated that lentil had Pb accumulation capacity and can be considered a Pb accumulator plant, elsewhere, roots accumulated more Pb than shoots, and the inoculation decreased the Pb up take by the plants, suggesting that this symbiosis should be investigated for use in phytostabilization of Pb-contaminated soils. At the same time, a modulation in the antioxidant enzyme activity and a specific duration was required for the induction of the superoxide dismutase (SOD), peroxidase (POX), and ascorbate peroxidase (APX) response and to adapt to Pb stress. These results suggested that these enzymes may be involved in the main mechanism of antioxidative defense in lentil exposed to Pb oxidative stress.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Chumbo/metabolismo , Chumbo/farmacologia , Lens (Planta)/microbiologia , Poluentes do Solo/metabolismo , Agrobacterium tumefaciens/isolamento & purificação , Biodegradação Ambiental , Biomassa , Cádmio/metabolismo , Farmacorresistência Bacteriana , Hidroponia , Lens (Planta)/enzimologia , Lens (Planta)/crescimento & desenvolvimento , Lens (Planta)/metabolismo , Estresse Oxidativo , Peroxidases/metabolismo , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/microbiologia , Pseudomonas/isolamento & purificação , Rahnella/isolamento & purificação , Rhizobium/isolamento & purificação , Nódulos Radiculares de Plantas/química , Nódulos Radiculares de Plantas/enzimologia , Nódulos Radiculares de Plantas/microbiologia , Solo/química , Microbiologia do Solo , Superóxido Dismutase/metabolismo
4.
Plant Sci ; 205-206: 29-37, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23498860

RESUMO

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme in plant starch biosynthesis. It contains large (LS) and small (SS) subunits encoded by two different genes. In this study, we explored the transcriptional regulation of both the LS and SS subunits of AGPase in stem and leaf under different photoperiods length in lentil. To this end, we first isolated and characterized different isoforms of the LS and SS of lentil AGPase and then we performed quantitative real time PCR (qPCR) to see the effect of photoperiod length on the transcription of the AGPase isforms under the different photoperiod regimes in lentil. Analysis of the qPCR results revealed that the transcription of different isoforms of the LSs and the SSs of lentil AGPase are differentially regulated when photoperiod shifted from long-day to short-day in stem and leaves. While transcript levels of LS1 and SS2 in leaf significantly decreased, overall transcript levels of SS1 increased in short-day regime. Our results indicated that day length affects the transcription of lentil AGPase isoforms differentially in stems and leaves most likely to supply carbon from the stem to other tissues to regulate carbon metabolism under short-day conditions.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Glucose-1-Fosfato Adenililtransferase/genética , Lens (Planta)/enzimologia , Fotoperíodo , Sequência de Bases , Clonagem Molecular , Glucose-1-Fosfato Adenililtransferase/isolamento & purificação , Glucose-1-Fosfato Adenililtransferase/metabolismo , Isoenzimas , Cinética , Lens (Planta)/genética , Lens (Planta)/efeitos da radiação , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/efeitos da radiação , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/efeitos da radiação , Sementes/enzimologia , Sementes/genética , Sementes/efeitos da radiação , Amido/metabolismo
5.
Biochem Cell Biol ; 91(2): 95-101, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23527638

RESUMO

In plants, cystathionine γ-synthase (CGS) and threonine synthase (TS) compete for the branch-point metabolite O-phospho-L-homoserine. These enzymes are potential targets for metabolic engineering studies, aiming to alter the flux through the competing methionine and threonine biosynthetic pathways, with the goal of increasing methionine production. Although CGS and TS have been characterized in the model organisms Escherichia coli and Arabidopsis thaliana, little information is available on these enzymes in other, particularly plant, species. The functional CGS and TS coding sequences from the grain legumes Cicer arietinum (chickpea) and Lens culinaris (lentil) identified in this study share approximately 80% amino acid sequence identity with the corresponding sequences from Glycine max. At least 7 active-site residues of grain legume CGS and TS are conserved in the model bacterial enzymes, including the catalytic base. Putative processing sites that remove the targeting sequence and result in functional TS were identified in the target species.


Assuntos
Carbono-Oxigênio Liases/genética , Cicer/genética , Regulação da Expressão Gênica de Plantas , Lens (Planta)/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Carbono-Oxigênio Liases/metabolismo , Cicer/enzimologia , Sequência Conservada , Escherichia coli/enzimologia , Escherichia coli/genética , Lens (Planta)/enzimologia , Metionina/biossíntese , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Soja/enzimologia , Soja/genética , Treonina/biossíntese , Tabaco/enzimologia , Tabaco/genética
6.
J Agric Food Chem ; 60(19): 4765-72, 2012 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-22534011

RESUMO

Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL(-1)) did not vary for at least 15 months when stored at 4 °C (k(inact) = 0.146 year(-1), t(1/2 inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k(inact) = 2.2 × 10(-3) day(-1), t(1/2 inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurified by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H(2)O(2)-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K(m)(appH(2)O(2)) = 1.87 mM; V(max)(appH(2)O(2)) = 6.4 mM min(-1); K(m)(app guaicol) = 32 mM; V(max)(app guaicol) = 9.1 mM min(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases. Finally, after the effectiveness of the crude extracts of LSP in oxidizing and removing from solution a series of last-generation dyes present in effluents from textile industries (1) had been checked, a steady-state kinetic study of the H(2)O(2)-mediated oxidation and decolorization of Green Domalan BL by the catalytic action of the lentil stubble extract was carried out, with the observation of the same apparent Michaelian kinetic behavior (K(m)(appGD) = 471 µM; V(max)(appGD)= 23 µM min(-1)). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.


Assuntos
Lens (Planta)/enzimologia , Peroxidase/química , Proteínas de Plantas/química , Agricultura , Estabilidade Enzimática , Resíduos Industriais/análise , Cinética , Lens (Planta)/química , Peroxidase/isolamento & purificação , Peroxidase/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo
7.
Indian J Biochem Biophys ; 46(5): 366-70, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20027865

RESUMO

Alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified (26-fold) from the germinating seeds of lentil (Lens culinaris) by affinity precipitation with alginate. The purified enzyme gave a single band corresponding to molecular mass of 40 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were determined as 40 degrees C and 5.5, respectively. The enzyme was very stable at a temperature range of 4-65 degrees C and at a pH range of 4-7. The values of kinetic constants Km and Vmax using p-nitrophenyl-alpha-D-galactopyranoside (PNPG) as substrate were 0.191 mM and 0.73 U, respectively. Results suggest that affinity precipitation is an attractive process for the purification of alpha-galactosidase.


Assuntos
Alginatos/química , Lens (Planta)/enzimologia , alfa-Galactosidase/química , alfa-Galactosidase/isolamento & purificação , Precipitação Química , Germinação , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Concentração de Íons de Hidrogênio , Lens (Planta)/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Temperatura Ambiente , Fatores de Tempo , alfa-Galactosidase/metabolismo
8.
Biosci Biotechnol Biochem ; 72(1): 29-36, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18175931

RESUMO

The electrochemical behavior of redox centers in the active site of amine oxidases from lentil seedlings and Euphorbia characias latex was investigated using a mercury film electrode. Tyrosine-derived 6-hydroxydopa quinone (TPQ) and copper ions in the active site are redox centers of these amine oxidases. The enzymes undergo two reduction processes at negative potentials related to the reduction of the TPQ cofactor to the corresponding hydroquinones and the reduction of copper ions, (Cu(II)-->Cu(I)). Copper depleted enzymes, prepared by reduction with dithionite followed by dialysis against cyanide, undergo only one reduction process. Nyquist diagrams, recorded at potentials corresponding to the reduction of cofactors as dc-offset, represent charge transfer impedance followed by a Warburg-type line at low frequencies, indicating the occurrence of a diffusion controlled process in the rate-limiting step of the reduction process.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Euphorbia/enzimologia , Lens (Planta)/enzimologia , Plântula/enzimologia , Amina Oxidase (contendo Cobre)/química , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cobre/metabolismo , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/análise , Eletroquímica , Eletrodos , Cinética , Dados de Sequência Molecular , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo
9.
Biosci Biotechnol Biochem ; 71(7): 1644-9, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17617729

RESUMO

The relationships between the structural and energetic domains of lentil seedling amine oxidase (LSAO) were investigated using modifiers that target the active site and the carbohydrate moiety of the enzyme. An irreversible inhibitor, aminoguanidine, specifically modified the active site of the lentil enzyme, whereas sodium metaperiodate cleaves carbohydrate moieties covalently bound to the native enzyme. Differential scanning calorimetry (DSC) measurements were made on the modified LSAOs. Deconvolution of the reversible thermal DSC profiles of the modified enzyme gave three subpeaks (energetic domains), each of which was assigned to one of the three structural domains of the native protein. Our results led us to conclude that deglycosylation of LSAO has no effect on thermal stability, whereas binding of the inhibitor imparts more stability to the enzyme.


Assuntos
Amina Oxidase (contendo Cobre)/química , Lens (Planta)/enzimologia , Plântula/enzimologia , Varredura Diferencial de Calorimetria , Desnaturação Proteica , Estrutura Terciária de Proteína
10.
FEBS J ; 274(10): 2585-95, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17433047

RESUMO

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.


Assuntos
Amina Oxidase (contendo Cobre)/química , Lisina/química , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Euphorbia/enzimologia , Rim/enzimologia , Cinética , Lens (Planta)/enzimologia , Ressonância Magnética Nuclear Biomolecular , Ervilhas/enzimologia , Alinhamento de Sequência , Suínos , Isótopos de Xenônio
11.
Biochimie ; 88(7): 827-35, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16519984

RESUMO

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.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Benzoquinonas/metabolismo , Cobre/química , Xenônio/química , Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/isolamento & purificação , Benzoquinonas/química , Catálise , Cromatografia Líquida de Alta Pressão , Lens (Planta)/enzimologia , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Estrutura Molecular , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Plântula/enzimologia , Espectrometria de Fluorescência/métodos , Espectrofotometria/métodos , Espectrofotometria Ultravioleta/métodos , Isótopos de Xenônio
12.
Protein J ; 24(3): 183-91, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16096724

RESUMO

The thermal stability of copper/quinone containing amine oxidases from Euphorbia characias latex (ELAO) and lentil seedlings (LSAO) was measured in 100 mM potassium phosphate buffer (pH 7.0) following changes in absorbance at 292 nm. ELAO was shown to be about 10 degrees C more stable than LSAO. The dissociative thermal inactivation of ELAO was studied using putrescine as substrate at different temperatures in the range 47-70 degrees C, and a "conformational lock" was developed using the theory pertaining to oligomeric enzyme. Moreover ELAO was shown to be more stable towards denaturants than LSAO, as confirmed by dodecyl trimethylammonium bromide denaturation curves. A comparison of the numbers of contact sites in inter-subunits of ELAO relative to LSAO led us to conclude that the higher stability of ELAO to temperature and towards denaturants was due to the presence of larger number of contact sites in the conformational lock of the enzyme. This study also gives a putative common mechanism for thermal inactivation of amine oxidases and explains the importance of C-terminal conserved amino acids residues in this class of enzymes.


Assuntos
Amina Oxidase (contendo Cobre)/química , Euphorbia/química , Temperatura Alta , Látex/metabolismo , Lens (Planta)/enzimologia , Conformação Proteica , Plântula/enzimologia , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Sequência de Aminoácidos , Animais , Estabilidade Enzimática , Lens (Planta)/anatomia & histologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Desnaturação Proteica , Putrescina/metabolismo , Alinhamento de Sequência
13.
Biol Chem ; 386(1): 25-31, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15843144

RESUMO

The reaction of NO-derivatized polyamines called "NONOates" with an amine oxidase from lentil seedlings was studied. 3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) and 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine (DPTA-NONOate) were found to be irreversible inactivators of the lentil enzyme. The spectrum of the protein was strongly affected in the course of reaction with both compounds, leading to the formation of a covalent adduct with a stable band at 334 nm. The corresponding amine compounds diethylentriamine (DETA) and norspermidine (DPTA) were substrates of the lentil enzyme that did not lead to enzyme inactivation. Diethylamine-NONOate, not containing amino groups, was found to be an irreversible inactivator of the amine oxidase only in the presence of a substrate. Since all NONOates spontaneously decompose in solution with release of NO, it seems as if the latter is responsible for the enzyme inhibition. The insensitivity of the native enzyme to NO suggested that this compound was unreactive toward both the cofactors, 6-hydroxydopa quinone (TPQ) and Cu(II), and thus a model for the irreversible inactivation could involve the attack by NO of the Cu(I)-semiquinolamine radical catalytic intermediate.


Assuntos
Amina Oxidase (contendo Cobre)/química , Benzoquinonas/química , Cobre/química , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/química , Lens (Planta)/enzimologia , Óxido Nítrico/química , Catálise , Radicais Livres/química , Estrutura Molecular , Oxirredução , Plântula/enzimologia , Fatores de Tempo
14.
Biol Chem ; 385(3-4): 323-9, 2004 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15134347

RESUMO

Copper amine oxidase from lentil (Lens esculenta) seedlings was shown to catalyze the oxidative deamination of tyramine and three similar aromatic monoamines, benzylamine, phenylethylamine and 4-methoxyphenylethylamine. Tyramine, an important plant intermediate, was found to be both a substrate and an irreversible inhibitor of the enzyme whereas the other amines were not inhibitory. In the course of tyramine oxidation the enzyme gradually became inactivated with the concomitant appearance of a new absorption at 560 nm due to the formation of a stable adduct. Inactivation took place only in the presence of oxygen and was probably due to the reaction of the enzyme with the oxidation product of tyramine, p-hydroxyphenylacetaldehyde. The kinetic data obtained in this study indicate that tyramine represents a new interesting type of physiological mechanism-based inhibitor for plant copper amine oxidases.


Assuntos
Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Di-Hidroxifenilalanina/análogos & derivados , Lens (Planta)/enzimologia , Tiramina/farmacologia , Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/isolamento & purificação , Di-Hidroxifenilalanina/antagonistas & inibidores , Inibidores Enzimáticos/isolamento & purificação , Extratos Vegetais/antagonistas & inibidores , Extratos Vegetais/isolamento & purificação , Plântula , Tiramina/química , Tiramina/isolamento & purificação
15.
J Biochem Mol Biol ; 36(2): 167-72, 2003 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-12689514

RESUMO

The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.


Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/metabolismo , Lens (Planta)/enzimologia , Temperatura Ambiente , Amina Oxidase (contendo Cobre)/isolamento & purificação , Dimerização , Ativação Enzimática , Cinética , Conformação Proteica , Desnaturação Proteica , Plântula/enzimologia , Termodinâmica
16.
J Protein Chem ; 20(5): 405-11, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11732692

RESUMO

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).


Assuntos
Amina Oxidase (contendo Cobre)/química , Lens (Planta)/enzimologia , Amina Oxidase (contendo Cobre)/isolamento & purificação , Sequência de Aminoácidos , Calorimetria , Metabolismo Energético , Ativação Enzimática , Dados de Sequência Molecular , Alinhamento de Sequência , Temperatura Ambiente
17.
Planta ; 214(1): 37-45, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11762169

RESUMO

Copper-containing amine oxidase (CuAO) has been proposed to play a role in H2O2 production in plant cell walls during cell development and in response to pathogen attack. We have compared the localisation of CuAO in pea (Pisum sativum L.), lentil (Lens culinaris M.) and chick pea (Cicer arietinum L.) grown under different light conditions, using both immuno- and histochemical techniques. The enzyme was detected by indirect immunofluorescence in the cell walls of parenchyma tissues of etiolated pea and lentil plants and was particularly abundant at intercellular spaces. Upon de-etiolation, CuAO largely disappeared from cortical cell walls except in the region of intercellular spaces. In the apical internode of light-grown seedlings, CuAO occurred mainly in cortical cell walls and, to some extent, in cell walls of xylem vessels. In both the elongation zone and mature regions of roots, CuAO was restricted to cortical cell walls and some cell junctions close to the meristem. Extensin epitopes co-localised to intercellular spaces of the cortex in de-etiolated pea, indicating that CuAO may have a role in cell wall strengthening at intercellular spaces. In chick pea, the localisation of the enzyme varied between different cultivars that have differing susceptibility to the fungus Ascochyta rabiei. In a susceptible cultivar Calia, immunogold labelling localised CuAO to cell walls of the cortex, as in lentil and pea, while in a resistant cultivar Sultano, it was most abundant in xylem vessels and, in light-grown plants, in the epidermis. These expression patterns are discussed with regard to the possible functions of amine oxidase in cell growth, cell differentiation and pathogen resistance.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Parede Celular/enzimologia , Fabaceae/enzimologia , Peróxido de Hidrogênio/metabolismo , Estruturas Vegetais/enzimologia , Amina Oxidase (contendo Cobre)/imunologia , Amina Oxidase (contendo Cobre)/efeitos da radiação , Anticorpos Monoclonais/imunologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Parede Celular/efeitos da radiação , Cicer/citologia , Cicer/enzimologia , Cicer/crescimento & desenvolvimento , Epitopos , Fabaceae/citologia , Fabaceae/crescimento & desenvolvimento , Glicoproteínas/metabolismo , Imunidade Inata , Imuno-Histoquímica , Lens (Planta)/citologia , Lens (Planta)/enzimologia , Lens (Planta)/crescimento & desenvolvimento , Luz , Ervilhas/citologia , Ervilhas/enzimologia , Ervilhas/crescimento & desenvolvimento , Doenças das Plantas , Proteínas de Plantas/metabolismo , Estruturas Vegetais/citologia , Estruturas Vegetais/efeitos da radiação , Especificidade da Espécie
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