RESUMO
Proteins from Melipona beecheii honey were purified by concanavalin A (conA) affinity chromatography and eluted with a stepwise glucose gradient into fractions named F2-F5. The conA-unbound fraction (F1) was further separated by molecular exclusion into fractions named MbF1-1,2 and MbF1-3. All fractions were evaluated for antibacterial activity against foodborne pathogens and antioxidant capacity. F1 fraction possessed highest antimicrobial activity against S. aureus, L. monocytogenes, S. Typhimurium, E. coli and P. aeruginosa with MIC's 1.4 ± 0.2, 15 ± 1, 39 ± 2, 1 ± 0.1, and 75 ± 2 µg/mL, respectively. F1, MbF1-1,2 and MbF1-3 had bactericidal effect except against P. aeruginosa. When the antioxidant capacity of the fractions was determined, F2 had the highest antioxidant activity measured by DPPH radical scavenging activity (IC50 = 2.4 ± 0.4 µg/µL) and reducing power of Fe(III) (IC50 = 1.8 ± 0.2 µg/µL). We provide evidence that M. beecheii honey proteins possess broad spectrum antibacterial and antioxidant activity, the latter probably through their reducing agent and free radical scavenger properties.
RESUMO
The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr â¼7.57 kDa and pI â¼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the â¼7.57 kDa polypeptide, immunodetected an â¼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the â¼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the â¼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the â¼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense.
Assuntos
Capsicum/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Capsicum/genética , Capsicum/crescimento & desenvolvimento , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Focalização Isoelétrica , Peso Molecular , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
A photosystem II component, the PsbO protein is essential for maximum rates of oxygen production during photosynthesis, and has been extensively characterized in plants and cyanobacteria but not in symbiotic dinoflagellates. Its close interaction with D1 protein has important environmental implications since D1 has been identified as the primary site of damage in endosymbiotic dinoflagellates after thermal stress. We identified and biochemically characterized the PsbO homolog from Symbiodinium kawagutii as a 28-kDa protein, and immunolocalized it to chloroplast membranes. Chloroplast association was further confirmed by western blot on photosynthetic membrane preparations. TX-114 phase partitioning, chromatography, and SDS-PAGE for single band separation and partial peptide sequencing yielded peptides identical or with high identity to PsbO from dinoflagellates. Analysis of a cDNA library revealed three genes differing by only one aminoacid residue in the in silico-translated ORFs despite greater differences at nucleotide level in the untranslated, putative regulatory sequences. The consensus full amino acid sequence displayed all the characteristic domains and features of PsbO from other sources, but changes in functionally critical, highly conserved motifs were detected. Our biochemical, molecular, and immunolocalization data led to the conclusion that the 28-kDa protein from S. kawagutii is the PsbO homolog, thereby named SkPsbO. We discuss the implications of critical amino acid substitutions for a putative regulatory role of this protein.
Assuntos
Dinoflagellida/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Cloroplastos/metabolismo , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Dados de Sequência Molecular , Complexo de Proteína do Fotossistema II/metabolismo , Filogenia , Proteínas de Plantas/imunologia , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Homologia de Sequência de AminoácidosRESUMO
RACK1 is a scaffold protein with the ability to interact in a regulated manner with a diverse number of ligands from distinct signal-transduction pathways. This assessment allowed us to infer that it may be involved in different processes such as nodulation. In a recent study we showed by silencing, that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development in Phaseolus vulgaris. On the other hand, we have also observed that its over-expression provokes a dramatic phenotype in: (a) seedlings that have been exposed to heat, in which systemic necrosis is induced; and (b) in Agrobacterium rhizogenes-transformed roots, where nodulation is strongly inhibited and nodules show early senescent symptoms. These findings indicate that PvRACK1 may be an integrator of diverse signal-transduction pathways in processes as varied as nodulation, cell expansion, heat stress responses, and systemic activation of necrosis.
Assuntos
Fixação de Nitrogênio/fisiologia , Peptídeos/metabolismo , Phaseolus/crescimento & desenvolvimento , Microscopia Eletrônica , Peptídeos/fisiologia , Phaseolus/fisiologia , Receptores de Quinase C AtivadaRESUMO
Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar ß-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.
Assuntos
Phaseolus/fisiologia , Nodulação/genética , Raízes de Plantas/crescimento & desenvolvimento , Receptores de Superfície Celular/metabolismo , Rhizobium tropici/fisiologia , Membrana Celular/ultraestrutura , Proliferação de Células , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Morfogênese , Phaseolus/genética , Phaseolus/crescimento & desenvolvimento , Phaseolus/microbiologia , Fenótipo , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteína Quinase C/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA de Plantas/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhizobium tropici/genética , Rhizobium tropici/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia , Transdução de SinaisRESUMO
Partial peptide sequence of a 36 kDa protein from common bean embryo axes showed 100% identity with a reported beta-subunit of a heterotrimeric G protein from soybean. Analysis of the full sequence showed 96.6% identity with the reported soybean G(beta)-subunit, 86% with RACK1B and C from Arabidopsis and 66% with human and mouse RACK1, at the amino acid level. In addition, it showed 85.5, 85 and 83% identities with arcA from Solanum lycopersicum, Arabidopsis (RACK1A) and Nicotiana tabacum, respectively. The amino acid sequence displayed seven WD40 domains and two sites for activated protein kinase C binding. The protein showed a constant expression level but the mRNA had a maximum at 32 h post-imbibition. Western immunoblotting showed the protein in vegetative plant tissues, and in both microsomal and soluble fractions from embryo axes. Synthetic auxin treatment during germination delayed the peak of RACK1 mRNA expression to 48 h but did not affect the protein expression level while the polar auxin transport inhibitor, naphtylphtalamic acid had no effect on either mRNA or protein expression levels. Southern blot and genomic DNA amplification revealed a small gene family with at least one member without introns in the genome. Thus, the RACK1/arcA homolog from common bean has the following features: (1) it is highly conserved; (2) it is both soluble and insoluble within the embryo axis; (3) it is encoded by a small gene family; (4) its mRNA has a peak of expression at the time point of germination stop and (5) its expression is only slightly affected by auxin but unaffected by an auxin transport blocker.
Assuntos
Germinação , Phaseolus/genética , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Clonagem Molecular , DNA de Plantas/genética , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica de Plantas , Humanos , Ácidos Indolacéticos/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Neuropeptídeos/genética , Phaseolus/embriologia , Phaseolus/metabolismo , Filogenia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Western blot transfer buffer was modified to substitute the acute poison methanol, with the common rubbing alcohol, isopropanol in concentrations of as low as 5 % for protein electrotransfer. Commercially available molecular weight markers and rabbit serum were run on polyacrylamide gels and shown to be transferred adequately to both nitrocellulose and polyvinylidene difluoride membranes under either wet or semi-dry conditions with similar results in all cases. This procedure was successfully used for immunodetection of the rabbit IgG heavy chain from serum. Therefore, this represents a good alternative for less toxic and environmentally friendly conditions for western immunoblotting of proteins.
Assuntos
Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Imunoglobulina G/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , 2-Propanol , Animais , Soluções Tampão , CoelhosRESUMO
The microfilament network of cultured Glycine max cells (SB-1 line), and protoplasts was visualized with rhodamine-phalloidin under conditions that lysed the protoplast and changed the cell shape. The whole cell had the typical microfilament distribution of a "cage" around the nucleus, from which the large subcortical cables and transvacuolar strands radiated towards the cortex until it reached the cortical microfilament network. Upon cell wall removal, the network conserved its compartmentalization. Thus, the redistribution of the shape where the vacuole becomes a central entity, made the cytoplasm displace peripherally, but the network distribution was conserved. When protoplasts were lysed in a low osmotic medium, the vacuoles were gradually released intact. Under these conditions, the F-actin staining remained within the ghost of the cell, but none was detected in either emerging or almost completely released vacuoles. Most of the released F-actin was found in debris from the cell lysate in the form of microfilaments. When the ghosts were constrained in a coverslip with an air bubble, the shape of the ghost changed accordingly, but the microfilament network distribution remained constant. These results provide further evidence that the vacuole of plant cells does not have detectable associated F-actin. In addition, we demonstrate that the actin microfilament network is a moldable entity that can change its shape but keeps its distribution under constant conditions, in these cultured cells.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Glycine max/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Corantes Fluorescentes/farmacologia , Microscopia Eletrônica , Faloidina/química , Faloidina/farmacologia , Ligação Proteica , Protoplastos/metabolismo , Coelhos , Rodaminas/química , Rodaminas/farmacologia , Vacúolos/químicaRESUMO
Three lipoxygenase isoforms were isolated from Glycine max embryo axes. A number of proteins around 97 kDa cross-reacted with several anti-actin and anti-myosin antibodies and these were used to follow their purification through gel filtration, hydroxyapatite and anion exchange columns. The 97-kDa cross-reactive material eluted in the unbound fractions of the last anion exchange column, and displayed two components of pI's 6.2 and 6.3. Further phase partition of this fraction in TX-114 yielded a hydrophobic 97 kDa protein. Additionally, a 95-kDa protein was retained and eluted from this last column. Partial peptide sequences indicated that the 95 kDa protein was soybean lipoxygenase-1, the first 97 kDa protein was lypoxygenase-3, and the hydrophobic 97 kDa protein was lipoxygenase-2. Several possible reasons for the cross-reactivity with the antibodies are discussed. To our knowledge, this is the first example of individual lipoxygenase isoforms isolated from soybean embryo axes.
Assuntos
Glycine max/enzimologia , Lipoxigenase/isolamento & purificação , Actinas/imunologia , Sequência de Aminoácidos , Anticorpos/imunologia , Western Blotting , Reações Cruzadas , Germinação , Isoenzimas/química , Isoenzimas/imunologia , Isoenzimas/isolamento & purificação , Lipoxigenase/química , Lipoxigenase/imunologia , Dados de Sequência Molecular , Miosinas/imunologia , Sementes/enzimologia , Glycine max/embriologiaRESUMO
While studying the behavior of profilin from Phaseolus vulgaris seeds under native conditions, a high molecular weight species suggesting a complex of profilin and associated proteins was observed by Western immunoblotting. This putative complex was also observed when enzyme-linked secondary antibodies alone were used, and this apparently resulted from antibody association, through its glycosyl moieties, with the endogenous carbohydrate-binding activity from the seed extracts. This endogenous activity corresponded to that of purified phytohemagglutinin (PHA). In addition, the P. vulgaris lectin activity was very stable and was observed when the extracts were pretreated with varying concentrations of sodium dodecyl sulfate, Triton X-100, urea and beta-mercaptoethanol, or when membrane blots were boiled in water before incubation with antibody. The activity was abolished only if the membrane was boiled in 1% sodium dodecyl sulfate. This finding could also be useful to implement assays for carbohydrate-binding activity from cell or tissue extracts using different visualizable reagents bearing particular glycosyl moieties.