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1.
Artigo em Inglês | MEDLINE | ID: mdl-31812006

RESUMO

Concanavalin A is a representative of the plant protein group known as lectins. Many lectin proteins have useful characteristics for studies on cell division and cell surfaces. In this study, a new adsorbent for the specific separation of Concanavalin A was prepared by applying a silica particle surface imprinting method. First, silica particles were activated via acidic treatment, and then, 3-methacryloyloxypropyl trimethoxysilane (MPTMS) was used for modification. For the preparation of Concanavalin A surface-imprinted silica particles (Con A-MISPs), N-methacryloyl-l-histidine methyl ester (MAH) was used as a functional monomer. The silica particles were characterized using a Zetasizer, scanning electron microscopy equipment (SEM), and Fourier transform infrared spectroscopy (FTIR). The effects of parameters such as the pH, initial concentration of Concanavalin A, and temperature on the adsorption of Concanavalin A were determined. The maximum Concanavalin A adsorption onto Con A-MISPs was observed to be 305.2 mg/g at a pH of 6. The reusability of the Con A-MISPs was approximately 93.5%. The non-imprinted silica particles (NISPs) were prepared in the same manner without Concanavalin A to compare the surface imprinting factor. Selective binding studies were carried out with lysozyme and hemoglobin molecules. The selectivity of the Con A-MISPs was also investigated by isolating Concanavalin A from Canavalia ensiformis. The purity of the Concanavalin A was shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).


Assuntos
Canavalia/química , Concanavalina A/isolamento & purificação , Impressão Molecular/métodos , Dióxido de Silício/química , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Temperatura
2.
Biosens Bioelectron ; 124-125: 59-65, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30343157

RESUMO

An electrochemiluminescence (ECL) analytical platform was initially proposed based on the electrochemiluminescence resonance energy transfer (ECL-RET) mechanism for ultrasensitive detection of Concanavalin A (Con A). In this protocol, the glucose functionalized carboxylic g-C3N4 nanosheets (g-C3N4-COOH@Glu) and MnO2 nanoparticles covered carboxylic multi-wall carbon nanotubes (BSA@MnO2-MWCNTs-COOH@Glu) were synthesized and acted as ECL-RET electron donor and acceptor, respectively. Herein, glucose was served as the recognition element for binding Con A and MWCNTs was utilized as the carrier materials for loading MnO2. When the quenching probe BSA@MnO2-MWCNTs-COOH@Glu was incubated onto the modified electrodes via the specific carbohydrate-Con A interaction, the ECL signals of g-C3N4-COOH@Glu which used S2O82- as its coreactant have drastically declined. Under optimum conditions, this biosensor performed a sensitive detection of the Con A ranging from 1 × 10-5 to 1 × 104 ng/mL with a detection limit of 2.2 fg/mL (S/N = 3). Moreover, favorable analytical outcomes for detecion Con A in actual serum samples were obtained, exhibiting huge applications in clinical diagnosis of this assay.


Assuntos
Técnicas Biossensoriais/métodos , Concanavalina A/isolamento & purificação , Técnicas Eletroquímicas/métodos , Nanopartículas/química , Concanavalina A/química , Transferência de Energia , Hipersensibilidade , Limite de Detecção , Medições Luminescentes , Compostos de Manganês/química , Nanotubos de Carbono/química
3.
Int J Mol Sci ; 20(1)2018 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-30577614

RESUMO

Lectins are a widely studied group of proteins capable of specific and reversible binding to carbohydrates. Undoubtedly, the best characterized are those extracted from plants of the Leguminosae family. Inside this group of proteins, those from the Diocleinae subtribe have attracted attention, in particular Concanavalin A (ConA), the best-studied lectin of the group. Diocleinae lectins, also called ConA-like lectins, present a high similarity of sequence and three-dimensional structure and are known to present inflammatory, vasoactive, antibiotic, immunomodulatory and antitumor activities, among others. This high similarity of lectins inside the ConA-like group makes it possible to use them to study structure/biological activity relationships by the variability of both carbohydrate specificity and biological activities results. It is in this context the following review aims to summarize the most recent data on the biochemical and structural properties, as well as biological activities, of ConA-like lectins and the use of these lectins as models to study structure/biological activity relationships.


Assuntos
Concanavalina A/química , Concanavalina A/farmacologia , Lectinas/química , Lectinas/farmacologia , Carboidratos/química , Fenômenos Químicos , Concanavalina A/genética , Concanavalina A/isolamento & purificação , Mediadores da Inflamação/química , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Lectinas/genética , Lectinas/isolamento & purificação , Relação Estrutura-Atividade
4.
Int J Biol Macromol ; 120(Pt A): 566-577, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30145160

RESUMO

Plant lectins have been studied owing to their structural properties and biological effects that include agglutinating activity, antidepressant-like effect and antitumor property. The results from this work showed the effects of the lectin extracted from the Dioclea violacea plant (DVL) on the C6 rat glioma cell line. DVL treatment was able to induce caspase-3 activation, apoptotic cell death and cellular membrane damage. Furthermore, DVL decreased mitochondrial membrane potential and increased the number of acidic vesicles and cleavage of LC3, indicating activation of autophagic processes. DVL also significantly inhibited cell migration. Compared to ConA, a well-studied lectin extracted from Canavalia ensiformes seeds, some effects of DVL were more potent, including decreasing C6 glioma cell viability and migration ability. Taken together, the results suggest that DVL can induce glioma cell death, autophagy and inhibition of cell migration, displaying potential anti-glioma activity.


Assuntos
Autofagia/efeitos dos fármacos , Dioclea/química , Expressão Gênica/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/genética , Canavalia/química , Caspase 3/genética , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Concanavalina A/isolamento & purificação , Concanavalina A/farmacologia , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Lectinas de Plantas/isolamento & purificação , Ratos
5.
Biosens Bioelectron ; 104: 27-31, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29306029

RESUMO

A novel method for study the interaction between carbohydrate and lectin was developed in homogenous solution. DNA was introduced to the recognition process of carbohydrate with protein. Due to the versatile detection mode of DNA, the detection efficiency for carbohydrate and protein was highly promoted. Herein, two kinds of mannose-DNA conjugates were synthesized. One of which was used to prepare DNA-templated silver nanocluster (AgNCs) and the other one contained guanine-rich DNA sequences. When the mannose of two conjugates binding to lectin Concanavalin A (Con A), great enhancement on fluorescence intensity was obtained due to the proximity ligation of the DNA-templated Ag NCs with guanine-rich DNA sequences. Hence Con A can be quantified conveniently in homogenous solution using Ag NCs with low toxicity and tunable fluorescence. Moreover, carbohydrate functionalized DNA-templated AgNCs was also utilized for cancer cell imaging based on the recognition of mannose with mannose receptor on cancer cell MDA-MB-231.


Assuntos
Técnicas Biossensoriais , Concanavalina A/isolamento & purificação , Nanopartículas Metálicas/química , Imagem Óptica , Sequência de Bases/genética , Concanavalina A/química , Sondas de DNA/química , Fluorescência , Humanos , Nanoestruturas/química
6.
J Microbiol Biotechnol ; 27(12): 2241-2244, 2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29025256

RESUMO

The structure of concanavalin A (ConA) has been studied intensively owing to its specific interactions with carbohydrates and its heterometal (Ca²âº and Mn²âº) coordination. Most structures from X-ray crystallography have shown ConA as a dimer or tetramer, because the complex formation requires specific crystallization conditions. Here, we reported the monomeric structure of ConA with a resolution of 1.6 Å, which revealed that metal coordination could trigger sugar-binding ability. The calcium coordination residue, Asn14, changed the orientation of carbohydrate-binding residues and biophysical details, including structural information, providing valuable clues for the development and application of detection kits using ConA.


Assuntos
Canavalia/química , Concanavalina A/química , Concanavalina A/isolamento & purificação , Sítios de Ligação , Cálcio/química , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Lectinas , Substâncias Macromoleculares , Manganês/química , Modelos Moleculares , Conformação Proteica , Açúcares/metabolismo
7.
J Chromatogr A ; 1508: 33-41, 2017 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-28602504

RESUMO

This research article reports the coating of large pore silica microparticles with a maltose layer to which bioaffinity ligands were attached via reductive amination reaction between the aldehyde activated maltose and the amino groups of the bioaffinity ligands. This was achieved first by the periodate oxidation of the maltose-silica (MALT-silica) yielding pairs of aldehyde groups at each monosaccharide ring. These di-aldehyde functionalities were then reacted with the primary amino groups of protein bio-affinity ligands and eventually formed Schiff bases (i.e., aldimines) which were reduced using the mild reducing agent sodium cyanoborohydride to form stable amine linkages between the immobilized protein ligands and the maltose layer. Anti-human serum albumin antibody (aHSA), anti-human serum transferrin antibody (aTf) and concanavalin A (Con A) were the bio-affinity ligands immobilized onto the MALT-silica and were evaluated in high performance affinity chromatography (HPAC), namely immunoaffinity chromatography (IAC) and lectin affinity chromatography (LAC). Our initial studies reported here revealed zero or reduced nonspecific interactions with the two immunoaffinity sorbents (i.e., aHSA-MALT-silica and aTf-MALT-silica) and the lectin affinity sorbent (i.e., Con A-MALT-silica). The absence of nonspecific interactions is attributed to the hydrophilicity of the maltose layer and its shielding effect of the residual silanols (i.e., unreacted silanols) on the silica surface. Conversely, the IAC and LAC sorbents exhibited specific interactions with the target biomolecules, namely human serum albumin (HSA) and transferrin (Tf) in the case of aHSA-MALT-silica and aTf-MALT-silica columns, respectively, and glycoproteins known for their affinity to Con A in the case of Con A-MALT-silica column.


Assuntos
Cromatografia de Afinidade/instrumentação , Maltose/química , Dióxido de Silício/química , Albuminas/química , Anticorpos/química , Anticorpos/isolamento & purificação , Cromatografia de Afinidade/métodos , Concanavalina A/química , Concanavalina A/isolamento & purificação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes
8.
Bioresour Technol ; 213: 257-261, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26923569

RESUMO

Jack bean (JB, Canavalia ensiformis) is the source of bio-based products, such as proteins and bio-sugars that contribute to modern molecular biology and biomedical research. In this study, the use of jack bean was evaluated as a source for concanavalin A (Con A) and bio-sugar production. A novel method for purifying Con A from JBs was successfully developed using a glucosylated magnetic nano matrix (GMNM) as a physical support, which facilitated easy separation and purification of Con A. In addition, the enzymatic conversion rate of 2% (w/v) Con A extracted residue to bio-sugar was 98.4%. Therefore, this new approach for the production of Con A and bio-sugar is potentially useful for obtaining bio-based products from jack bean.


Assuntos
Biotecnologia/métodos , Canavalia/metabolismo , Concanavalina A/isolamento & purificação , Biotecnologia/instrumentação , Carboidratos , Glicosilação , Monossacarídeos/metabolismo , Nanoestruturas
9.
Artigo em Inglês | MEDLINE | ID: mdl-26102634

RESUMO

Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A.


Assuntos
Aptâmeros de Nucleotídeos/química , Canavalia/química , Cromatografia de Afinidade/métodos , Concanavalina A/isolamento & purificação , Aptâmeros de Nucleotídeos/metabolismo , Concanavalina A/química , Concanavalina A/metabolismo , Cloreto de Sódio
10.
ACS Appl Mater Interfaces ; 6(15): 13234-41, 2014 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-25014128

RESUMO

Biosensors for the detection of proteins and bacteria have been developed using glycopolymer-immobilized metal mesh devices. The trimethoxysilane-containing glycopolymer was immobilized onto a metal mesh device using the silane coupling reaction. The surface shape and transmittance properties of the original metal mesh device were maintained following the immobilization of the glycopolymer. The mannose-binding protein (concanavalin A) could be detected at concentrations in the range of 10(-9) to 10(-6) mol L(-1) using the glycopolymer-immobilized metal mesh device sensor, whereas another protein (bovine serum albumin) was not detected. A detection limit of 1 ng mm(-2) was achieved for the amount of adsorbed concanavalin A. The glycopolymer-immobilized metal mesh device sensor could also detect bacteria as well as protein. The mannose-binding strain of Escherichia coli was specifically detected by the glycopolymer-immobilized metal mesh device sensor. The glycopolymer-immobilized metal mesh device could therefore be used as a label-free biosensor showing high levels of selectivity and sensitivity toward proteins and bacteria.


Assuntos
Bactérias/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Manosídeos/química , Metais/química , Proteínas/isolamento & purificação , Silanos/química , Aderência Bacteriana , Simulação por Computador , Concanavalina A/isolamento & purificação , Campos Eletromagnéticos , Escherichia coli/isolamento & purificação , Microscopia de Força Atômica , Espectroscopia Fotoeletrônica , Soroalbumina Bovina/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier , Coloração e Rotulagem , Propriedades de Superfície
11.
Biosens Bioelectron ; 59: 404-11, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24768820

RESUMO

There is no comparative data available on the binding constants of Concanavalin A (Con A) and glycogen and Con A-mannan using quartz crystal microbalance (QCM), cost and time efficient system for biosensor analysis. It is hypothesized that a QCM can be used in its flow injection mode to monitor the binding affinity of polysaccharides to an immobilized lectin, Con A. The biosensor is prepared by immobilizing Con A on a 5MHz gold crystal by carbodiimide crosslinking chemistry. The attachment efficiency is monitored by Fourier Transform Infrared Spectroscopy. Equilibrium association and dissociation constants describing Con A-polysaccharides interaction are determined in a saturation binding experiment, where increasing concentrations of polysaccharides are run on a Con A-immobilized gold crystal surface, and the frequency shifts recorded on the frequency counter. The molecular weights (MW) of glycogen from Oyster and mannan from Saccharomyces cerevisiae are determined by size exclusion chromatography. The MW for glycogen and mannan are 604±0.002 kDa and 54±0.002 kDa, respectively. The equilibrium association and dissociation constants for Con A-glycogen and Con A-mannan interactions are KA=3.93±0.7×10(6) M(-1)/KD=0.25±0.06 µM and (n=3), respectively. Their respective frequency and motional resistance shifts relationship (ΔF/ΔR) are 37.29±1.55 and 34.86±0.85 Hz/Ω (n=3), which support the validity of Sauerbrey׳s rigidity approximation. This work suggests that Con A-mannan complex could be potentially utilized for insulin delivery and the targeting of glucose-rich substances and glycoproteins when fast drug release is desired.


Assuntos
Concanavalina A/metabolismo , Glicogênio/metabolismo , Ouro/química , Mananas/metabolismo , Técnicas de Microbalança de Cristal de Quartzo , Técnicas Biossensoriais , Canavalia/química , Concanavalina A/isolamento & purificação , Ligação Proteica , Técnicas de Microbalança de Cristal de Quartzo/métodos
12.
Biosens Bioelectron ; 34(1): 202-7, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22387041

RESUMO

In this paper, a novel method for detecting concanavalin A (Con A) was developed based on lectin-carbohydrate biospecific interactions. Multi-wall carbon nanotube-polyaniline (MWNT-PANI) nanocomposites, synthesized by in situ polymerization, were chosen to immobilize d-glucose through the Schiff-base reaction. The immobilized D-glucose showed high binding sensitivity and excellent selectivity to its target lectin, Con A. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were applied to characterize the assembly process of the modified electrode. Due to the high affinity of Con A for D-glucose and high stability of the propounded sensing platform, the fabricated biosensor achieved ultrasensitive detection of Con A with good sensitivity, acceptable reproducibility and stability. The changes of response current were proportional to the Con A concentrations from 3.3 pM to 9.3 nM, with a detection limit of 1.0 pM. Therefore, the combination of MWNT-PANI nanocomposites and the special binding force between lectin and carbohydrate provides an efficient and promising platform for the fabrication of bioelectrochemical devices.


Assuntos
Técnicas Biossensoriais/métodos , Carboidratos/química , Concanavalina A/isolamento & purificação , Nanotubos de Carbono/química , Compostos de Anilina/química , Técnicas Biossensoriais/instrumentação , Concanavalina A/química , Limite de Detecção , Sensibilidade e Especificidade
13.
J Chromatogr A ; 1218(32): 5487-97, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21742336

RESUMO

A capillary-based model modified for characterization of monolithic cryogels is presented with key parameters like the pore size distribution, the tortuosity and the skeleton thickness employed for describing the porous structure characteristics of a cryogel matrix. Laminar flow, liquid dispersion and mass transfer in each capillary are considered and the model is solved numerically by the finite difference method. As examples, two poly(hydroxyethyl methacrylate) (pHEMA) based cryogel beds have been prepared by radical cryo-copolymerization of monomers and used to test the model. The axial dispersion behaviors, the pressure drop vs. flow rate performance as well as the non-adsorption breakthrough curves of different proteins, i.e., lysozyme, bovine serum albumin (BSA) and concanavalin A (Con A), at various flow velocities in the cryogel beds are measured experimentally. The lumped parameters in the model are determined by matching the model prediction with the experimental data. The results showed that for a given cryogel column, by using the model based on the physical properties of the cryogel (i.e., diameter, length, porosity, and permeability) together with the protein breakthrough curves one can obtain a reasonable estimate and detailed characterization of the porous structure properties of cryogel matrix, particularly regarding the number of capillaries, the capillary tortuousness, the pore size distribution and the skeleton thickness. The model is also effective with regards to predicting the flow performance and the non-adsorption breakthrough profiles of proteins at different flow velocities. It is thus expected to be applicable for characterizing the properties of cryogels and predicting the chromatographic performance under a given set of operating conditions.


Assuntos
Cromatografia Líquida/instrumentação , Concanavalina A/isolamento & purificação , Hidrogéis/química , Muramidase/isolamento & purificação , Soroalbumina Bovina/isolamento & purificação , Adsorção , Animais , Bovinos , Concanavalina A/química , Criogéis , Modelos Químicos , Muramidase/química , Porosidade , Soroalbumina Bovina/química
14.
J Phys Chem B ; 115(20): 6691-9, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21534632

RESUMO

In this work, triply responsive films with a specific binary architecture combining layer-by-layer assembly (LbL) and hydrogel polymerization were successfully prepared. First, concanavalin A (Con A) and dextran (Dex) were assembled into {Con A/Dex}(5) LbL layers on electrode surface by the lectin-sugar biospecific interaction between them. The poly(N,N-diethylacrylamide) (PDEA) hydrogels with entrapped horseradish peroxidase (HRP) were then synthesized by polymerization on the surface of LbL inner layers, forming {Con A/Dex}(5)-(PDEA-HRP) films. The films demonstrated reversible pH-, thermo-, and salt-responsive on-off behavior toward electroactive probe Fe(CN)(6)(3-) in its cyclic voltammetric responses. This multiple stimuli-responsive films could be further used to realize triply switchable electrochemical reduction of H(2)O(2) catalyzed by HRP immobilized in the films and mediated by Fe(CN)(6)(3-) in solution. The responsive mechanism of the films was explored and discussed. The pH-sensitive property of the system was attributed to the electrostatic interaction between the {Con A/Dex}(5) inner layers and the probe at different pH, and the thermo- and salt-responsive behaviors should be ascribed to the structure change of PDEA hydrogels for the PDEA-HRP outermost layers under different conditions. The concept of binary architecture was also used to fabricate {Con A/Dex}(5)-(PDEA-GOD) films on electrodes, where GOD = glucose oxidase, which was applied to realize the triply switchable bioelectrocatalysis of glucose by GOD in the films with ferrocenedicarboxylic acid as the mediator in solution. This film system with the unique binary architecture may establish a foundation for fabricating a novel type of multicontrollable biosensors based on bioelectrocatalysis with immobilized enzymes.


Assuntos
Acrilamidas/química , Armoracia/enzimologia , Técnicas Biossensoriais/métodos , Canavalia/química , Concanavalina A/metabolismo , Dextranos/química , Peroxidase do Rábano Silvestre/metabolismo , Polímeros/química , Concanavalina A/isolamento & purificação , Técnicas Eletroquímicas/métodos , Eletrodos , Glucose/metabolismo , Glucose Oxidase/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/metabolismo , Oxirredução , Polimerização , Temperatura
15.
Chem Commun (Camb) ; 47(13): 3930-2, 2011 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-21350742

RESUMO

We present a novel approach to constructing glycosylated surface for microporous membrane. Carbohydrate derivative can be facilely bound onto the alkyne-modified membrane surface via thiol-yne click chemistry. The glycosylated membrane surface shows an excellent affinity adsorption to lectin on the basis of carbohydrate-protein recognition.


Assuntos
Alquinos/química , Química Click/métodos , Concanavalina A/isolamento & purificação , Membranas Artificiais , Compostos de Sulfidrila/química , Adsorção , Glicosilação , Porosidade , Sensibilidade e Especificidade , Propriedades de Superfície
16.
J Chromatogr B Analyt Technol Biomed Life Sci ; 879(5-6): 457-60, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21256095

RESUMO

A PEG/citrate aqueous two-phase system was tested in the partition of commercial Concanavalin A (Con A) and subsequently applied to the extraction and purification of Con A from the crude extract of Canavalia ensiformis seeds. Con A was successfully extracted to the bottom phase of a system composed of 22% (w/w) PEG8000 and 12% (w/w) citrate at pH 6.0. The obtained purification factor was 11.5 without any loss in the hemagglutinating activity. The purity of extracted lectin was confirmed by SDS-PAGE analysis.


Assuntos
Canavalia/química , Fracionamento Químico/métodos , Ácido Cítrico/química , Concanavalina A/isolamento & purificação , Polietilenoglicóis/química , Análise de Variância , Concanavalina A/química , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Peso Molecular , Extratos Vegetais/química , Sementes/química
17.
EMBO J ; 29(19): 3318-29, 2010 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-20802462

RESUMO

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady-state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1α and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1-deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1α, and mixed-disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI-family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1- and glutathione disulphide-mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.


Assuntos
Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Células Cultivadas , Concanavalina A/isolamento & purificação , Primers do DNA/genética , Densitometria , Glutationa/metabolismo , Humanos , Imunoprecipitação , Cinética , Oxirredução , Isomerases de Dissulfetos de Proteínas/metabolismo , Transfecção
18.
J Agric Food Chem ; 58(7): 4090-6, 2010 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-20201549

RESUMO

This study reports on the antifungal activity of Dgui, a ConA-like lectin from Dioclea guianensis seeds. Dgui inhibited conidial germination but not mycelial growth of Colletotrichum gloeosporioides. The lectins ConA and ConM from Canavalia ensiformis and Canavalia maritima, respectively, share high levels of amino acid sequence similarity (>84%) with Dgui and have the same specificity toward glucose/mannose but had no effect on the fungus. Fluorescence microscopy showed that both Dgui and ConM bind to C. gloeosporioides ungerminated conidia. However, Dgui did not bind to C. gloeosporioides germinated conidia and germ tubes and was not inhibitory to mycelial growth. Because only Dgui inhibited germination of the fungus, C. gloeosporioides conidia might have surface-specific germination targets recognized by Dgui but not by its homologues, ConM and ConA. Therefore, Dgui is a candidate for biotechnological approaches for improving the resistance of various nutritionally and commercially important crops that are affected by C. gloeosporioides.


Assuntos
Antifúngicos/farmacologia , Colletotrichum/efeitos dos fármacos , Concanavalina A/farmacologia , Dioclea/metabolismo , Lectinas/farmacologia , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Colletotrichum/crescimento & desenvolvimento , Concanavalina A/química , Concanavalina A/isolamento & purificação , Dioclea/química , Lectinas/química , Lectinas/economia , Lectinas/isolamento & purificação , Lectinas/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência
19.
J Biomed Biotechnol ; 2009: 179106, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19547713

RESUMO

A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.


Assuntos
Óxido Ferroso-Férrico/química , Frutanos/química , Lectinas de Plantas/isolamento & purificação , Canavalia/química , Cromatografia Líquida , Concanavalina A/isolamento & purificação , Fabaceae/química , Frutanos/metabolismo , Testes de Inibição da Hemaglutinação , Ressonância Magnética Nuclear Biomolecular , Lectinas de Plantas/metabolismo , Zymomonas/química
20.
Int J Biol Macromol ; 43(3): 279-82, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18640151

RESUMO

The water-soluble polysaccharide (CPP), with a molecular mass of 1.1x10(4) Da, was obtained from the roots of Codonopsis pilosula. Structure feature investigation by a combination of chemical and instrumental analysis revealed that CPP had a backbone consisting of (1-->3)-linked-beta-D-galactopyranosyl, (1-->2, 3)-linked-beta-D-galactopyranosyl and (1-->3)-linked-alpha-D-rhamnopyranosyl residues, which were branched with two glycosyl residues composed of alpha-L-arabinose-(1-->5)-alpha-L-arabinose(1-->linked residues at the O-2 position of galactosyl along the main chain in the ratio of 1:1:2:1:1. Preliminary immunological tests in vitro showed CPP could stimulate concanavalin A (ConA)- or lipopolysaccharide (LPS)-induced lymphocyte proliferation in a dose-dependent manner.


Assuntos
Codonopsis/química , Raízes de Plantas/química , Polissacarídeos/química , Polissacarídeos/imunologia , Água/química , Animais , Proliferação de Células/efeitos dos fármacos , Concanavalina A/química , Concanavalina A/imunologia , Concanavalina A/isolamento & purificação , Concanavalina A/farmacologia , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/farmacologia , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Solubilidade
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