RESUMEN
The behavior of human immunoglobulin G (IgG) and antigen-binding fragment (Fab fragment) adsorption onto phospho-l-tyrosine immobilized on agarose (P-Tyr-agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis-Tris, Tris-HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L-1 NaP buffer at pH 6.0. The capture of IgG by the P-Tyr-agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L-1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo-second-order model. The adsorption isotherm data were well described by the Langmuir-Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P-Tyr-agarose from digested human IgG in HEPES 25 mmol L-1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.
Asunto(s)
Cromatografía de Afinidad/métodos , Cromatografía en Agarosa/métodos , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Fosfotirosina/química , Adsorción , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Papaína/metabolismoRESUMEN
Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.
Asunto(s)
Inmunoprecipitación/métodos , Insulina/análisis , Islotes Pancreáticos/química , Proproteína Convertasa 2/análisis , Vesículas Secretoras/química , Especificidad de Anticuerpos , Cromatografía en Agarosa/métodos , Electroforesis/métodos , Glucosa/farmacología , Humanos , Concentración de Iones de Hidrógeno , Inmunoprecipitación/instrumentación , Inmunoadsorbentes , Insulina/biosíntesis , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Marcaje Isotópico/métodos , Metionina/análisis , Proproteína Convertasa 2/biosíntesis , Vesículas Secretoras/enzimología , Radioisótopos de Azufre/análisis , UreaRESUMEN
Los líquenes son los hongos que establecen una relación simbiótica con un alga o cianobacteria. En esta simbiosis se producen por parte del hongo, una serie de metabolitos secundarios conocidos como sustancias liquénicas; las cuales presentan una marcada actividad antibiótica. En Cuba no se tienen antecedentes sobre estudios de metabolitos liquénicos por lo que se propone; evaluar el efecto fungicida de extractos liquénicos producidos por especies cubanas así como identificar sus metabolitos. Se emplearon líquenes de diferentes zonas del país (Parmotrema dilatatum, P. tinctorum, P. praesorediosum P. cristiferum, Ramalina americana, Cladonia ceratophylla y Cladonia portentosa spp. pacífica), a los cuales se les extrajo con acetona, las sustancias liquénicas almacenadas en el talo. Los extractos fueron probados contra los hongos fitopatógenos Rhizoctonia solani y Phythophtora nicotianae; por el método de envenenamiento del medio de cultivo agar papa dextrosa a concentraciones de: 0,01 por ciento; 0,03 por ciento y 0,07 por ciento. Se utilizó un control negativo de dimetilsufóxido al 0,07 por ciento y se determinaron los porcentajes de inhibición, cuyos resultados fueron analizados estadísticamente. Los metabolitos secundarios presentes en los extractos se identificaron por cromatografía de capa fina (TLC). Exceptuando el extracto liquénico de P. cristiferum, todos los demás mostraron más de un 50 por ciento de inhibición del crecimiento de ambos hongos a la concentración de 0,07 por ciento, mientras que a las restantes concentraciones los valores fueron variados con diferencias significativas con respecto al control. Se lograron identificar tres metabolitos liquénicos: metil 2-O- metilmicrofilinato, 4-O-Demetilmicrofilinico y el ácido ramaniloico.
Lichens are fungi that establish a symbiotic relationship with an alga or cyanobacterium. This symbiosis produced by the fungus, a series of secondary metabolites known as lichen substances; which show a strong antibiotic activity. In Cuba there is no background on the studies above lichen metabolites so it is proposed; evaluate the fungicidal activity of lichen extracts produced by Cuban species and to identify metabolites. Lichens from different areas of the country (Parmotrema dilatatum, P. tinctorum, P.praesorediosum, P. cristiferum, Ramalina americana, Cladonia ceratophylla and Cladonia portentosa spp. pacífica), to which it extracted with acetone, the lichen substances stored in tallus. The extracts were tested against the fungal pathogens Rhizoctonia solani and Phythophtora nicotianae; poisoning by the method of culture medium potato dextrose agar at concentrations of 0.01 percent; 0.03 percent and 0.07 percent. A negative control to 0.07 percent dimethylsulfoxide was used and the percentage of inhibition, the results were analyzed statistically determined. Secondary metabolites present in the extracts were identified by thin layer chromatography (TLC). Except P. cristiferum lichen extract, all others showed more than 50 percent growth inhibition of both fungi at concentration of 0.07 percent, while the remaining concentrations were varied values with significant differences from the control. Was made to identify three lichen metabolites metilmicrofilinato 2 methyl-O, 4-ODemetilmicrofilinico and ramaniloico acid.
Asunto(s)
Antígenos Fúngicos/aislamiento & purificación , Antígenos Fúngicos/análisis , Líquenes/metabolismo , Líquenes/química , Acetona , Agar , Antifúngicos , Cuba , Medios de Cultivo , Cromatografía en Agarosa/métodos , Hongos/patogenicidad , IntoxicaciónRESUMEN
Recently, it has been demonstrated that fructose-2,6-bisphosphate (F2,6BP) protects skeletal muscle 6-phosphofructo-1-kinase (PFK) from thermal inactivation (50 degrees C) and against the deleterious effects of guanidinium hydrochloride (GdmCl). On the other hand, ATP, when added at its inhibitory concentrations, that is, >1 mM, enhanced either the thermal- or GdmCl-induced inactivation of PFK. Moreover, we concluded that these phenomena were probably due to the stabilization of PFK tetrameric structure by F2,6BP, and the dissociation of this structure into dimers induced by ATP. Aimed at elucidating the effects of F2,6BP and ATP on PFK at the structural and functional levels, the present work correlates the effects of these metabolites on the equilibrium between PFK dimers and tetramers to the regulation promoted on the enzyme catalytic activity. We show that ATP present a dual effect on PFK structure, favoring the formation of tetramer at stimulatory concentrations (up to 1 mM), and dissociating tetramers into dimers at inhibitory concentrations (>1 mM). Furthermore, F2,6BP counteracted this later ATP effect at either the structural or catalytic levels. Additionally, the effects of both F2,6BP or ATP on the equilibrium between PFK tetramers and dimers and on the enzyme activity presented a striking parallelism. Therefore, we concluded that modulation of PFK activity by ATP and F2,6BP is due to the effects of these ligands on PFK quaternary structure, altering the oligomeric equilibrium between PFK tetramers and dimers.
Asunto(s)
Adenosina Trifosfato/metabolismo , Fructosadifosfatos/metabolismo , Músculo Esquelético/metabolismo , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1/metabolismo , Estructura Cuaternaria de Proteína/genética , Cromatografía en Agarosa , Dimerización , CinéticaRESUMEN
A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.
Asunto(s)
Animales , Ratones , Coagulación Sanguínea , Bothrops , Coagulantes/aislamiento & purificación , Venenos de Crotálidos/enzimología , Serina Endopeptidasas/aislamiento & purificación , Secuencia de Aminoácidos , Antivenenos/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Cromatografía en Agarosa , Cromatografía por Intercambio Iónico , Costa Rica , Coagulantes/administración & dosificación , Coagulantes/farmacología , Evaluación Preclínica de Medicamentos , Fibrinógeno/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/farmacología , Mordeduras de Serpientes/fisiopatología , Trombina/químicaRESUMEN
Azospirillum sp promotes the growth of many important crop plants. We demonstrated lectin binding activity in outer-membrane protein extracts of A. brasilense Sp7 by hemagglutination assays. The lectin specifically recognised the exopolysaccharide (EPS) produced by aggregated cells. Affinity chromatography using EPS-Sepharose was used to identify a 67 kDa outer-membrane lectin (OML) that recognised a binding region in the extracellular polysaccharide. Results show the specific recognition and binding between EPS and OML. The potential relationship between cell-to-cell aggregation and the OML-EPS interaction is discussed.
Asunto(s)
Azospirillum brasilense/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Lectinas/metabolismo , Polisacáridos Bacterianos/metabolismo , Arabinosa/metabolismo , Adhesión Bacteriana , Cromatografía de Afinidad , Cromatografía en Agarosa , Pruebas de Inhibición de Hemaglutinación , Unión Proteica , Especificidad por SustratoRESUMEN
A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.
Asunto(s)
Coagulación Sanguínea , Bothrops , Coagulantes/aislamiento & purificación , Venenos de Crotálidos/enzimología , Serina Endopeptidasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antivenenos/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Cromatografía en Agarosa , Cromatografía por Intercambio Iónico , Coagulantes/administración & dosificación , Coagulantes/farmacología , Costa Rica , Evaluación Preclínica de Medicamentos , Fibrinógeno/metabolismo , Ratones , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/farmacología , Mordeduras de Serpientes/fisiopatología , Trombina/químicaRESUMEN
The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K (m) 0.28 and 0.22 mmol/L, with upsilon (lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.
Asunto(s)
Fosfatasa Alcalina/aislamiento & purificación , Proteínas Fúngicas/aislamiento & purificación , Rhizopus/enzimología , Fosfatasa Alcalina/metabolismo , Cationes/farmacología , Cromatografía en Agarosa , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Peso MolecularRESUMEN
This work investigated the potential use of an alternative adsorbent to anti-immunoglobulin E (IgE)-agarose for IgE selective adsorption therapy. A screening of several commercially available adsorbents (Concanavalin A, Lens culinaris[Lc], d-tryptophan, poly-l-lysine, and aminohexyl immobilized on agarose) was done through batch system assays, considering some criteria, such as adsorption capacity, selectivity, and biocompatibility. In the Lc-agarose adsorbent, total IgE, and specific IgE--for the airborne allergens Dermatophagoides pteronyssinus and Blomia tropicalis--were significantly better removed (63, 58, and 59%, respectively) than immunoglobulin G (19%), immunoglobulin A (33%), immunoglobulin M (9%), and albumin (18%). This adsorbent was packed into a column and the effect of superficial velocity, ratio of plasma volume to bed volume, number of perfusions, and temperature on IgE adsorption were evaluated. In vitro simulation of therapeutic adsorption (single perfusion) indicated that about 50% of total IgE could be eliminated.
Asunto(s)
Eliminación de Componentes Sanguíneos/métodos , Inmunoglobulina E/metabolismo , Inmunoadsorbentes/farmacocinética , Adsorción , Cromatografía en Agarosa/métodos , Convertasas de Complemento C3-C5/metabolismo , Circulación Extracorporea/métodos , Humanos , Hipersensibilidad/terapia , Técnicas de Inmunoadsorción , Inmunoadsorbentes/uso terapéutico , Ligandos , Lectinas de Plantas/farmacocinética , Lectinas de Plantas/uso terapéutico , Polilisina/análogos & derivados , Polilisina/farmacocinética , Polilisina/uso terapéutico , Sefarosa/análogos & derivados , Sefarosa/farmacocinética , Sefarosa/uso terapéutico , Triptófano/farmacocinética , Triptófano/uso terapéuticoRESUMEN
Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.
Asunto(s)
Venenos de Artrópodos/farmacología , Proteínas de la Matriz Extracelular/efectos de los fármacos , Hemolinfa/enzimología , Mariposas Nocturnas/enzimología , Serina Endopeptidasas/farmacología , Animales , Venenos de Artrópodos/aislamiento & purificación , Cromatografía en Agarosa , Fibronectinas/efectos de los fármacos , Hemorragia/inducido químicamente , Hemorragia/enzimología , Hemostasis/efectos de los fármacos , Hemostasis/fisiología , Humanos , Laminina/efectos de los fármacos , Serina Endopeptidasas/aislamiento & purificación , Vitronectina/efectos de los fármacosRESUMEN
Two isoforms of laccase produced from the culture supernatant of Pycnoporus sanguineus were partially purified by phenyl-Sepharose chromatography. Molecular masses of the enzymes were 80 kDa (Lac I) and 68 kDa (Lac II). Optimum activity of Lac I was at pH 4.8 and 30 degrees C, and Lac II was at pH 4.2 and 50 degrees C over 5 min reaction. The Km values of enzymes toward syringaldazine were 10 microM: (Lac I) and 8 microM: (Lac II). Sodium azide inhibited Lac I (85%) and Lac II (75%) activities.
Asunto(s)
Lacasa/biosíntesis , Polyporaceae/efectos de los fármacos , Polyporaceae/enzimología , Biotecnología , Cromatografía en Agarosa , Inducción Enzimática/efectos de los fármacos , Estabilidad de Enzimas , Hidrazonas/metabolismo , Concentración de Iones de Hidrógeno , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Lacasa/química , Lacasa/metabolismo , Peso Molecular , Especificidad por SustratoRESUMEN
Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.
Asunto(s)
Malato Deshidrogenasa/análisis , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía en Agarosa/métodos , Reacciones Cruzadas/inmunología , Citosol/enzimología , Regulación del Desarrollo de la Expresión Génica/genética , Genes Protozoarios/genética , Isoenzimas/análisis , Isoenzimas/inmunología , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/inmunología , Microcuerpos/enzimología , Microcuerpos/genética , Microcuerpos/inmunología , Mitocondrias/enzimología , Mitocondrias/genética , Mitocondrias/inmunología , Ácido Oxaloacético/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Filogenia , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia/métodos , Trypanosoma brucei brucei/inmunologíaRESUMEN
Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 Ag) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 Ag) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms (AU)
El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 Ag de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 Ag.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes (AU)
Asunto(s)
Animales , Masculino , Ratones , Conejos , Crotalus/inmunología , Crotoxina/inmunología , Fosfolipasas A/inmunología , Antivenenos/inmunología , Inmunoglobulina G/inmunología , Pruebas de Neutralización/métodos , Crotoxina/toxicidad , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/toxicidad , Antivenenos/biosíntesis , Antivenenos/farmacología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/farmacología , Bloqueo Neuromuscular , Immunoblotting , Inmunoelectroforesis , Ensayo de Inmunoadsorción Enzimática , Especificidad de Anticuerpos , Cromatografía en Agarosa , Tampones (Química) , Hemólisis/inmunología , Modelos Animales de EnfermedadRESUMEN
Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 Ag) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 Ag) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms (AU)
El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 Ag de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 Ag.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes (AU)
Asunto(s)
Animales , Masculino , Ratones , Conejos , Crotalus/inmunología , Crotoxina/inmunología , Fosfolipasas A/inmunología , Antivenenos/inmunología , Inmunoglobulina G/inmunología , Pruebas de Neutralización/métodos , Crotoxina/toxicidad , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/toxicidad , Antivenenos/biosíntesis , Antivenenos/farmacología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/farmacología , Bloqueo Neuromuscular , Immunoblotting , Inmunoelectroforesis , Ensayo de Inmunoadsorción Enzimática , Especificidad de Anticuerpos , Cromatografía en Agarosa , Tampones (Química) , Hemólisis/inmunología , Modelos Animales de EnfermedadRESUMEN
Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms
El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes
Asunto(s)
Animales , Masculino , Ratones , Conejos , Antivenenos/inmunología , Crotalus/inmunología , Crotoxina/inmunología , Inmunoglobulina G/inmunología , Pruebas de Neutralización/métodos , Fosfolipasas A/inmunología , Especificidad de Anticuerpos , Antivenenos/biosíntesis , Antivenenos/farmacología , Tampones (Química) , Cromatografía en Agarosa , Crotoxina/toxicidad , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hemólisis/inmunología , Immunoblotting , Inmunoelectroforesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/farmacología , Bloqueo Neuromuscular , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/toxicidadRESUMEN
In a chromatographic method modification intended to preserve protease activity in Bothrops erythromelas venom, 2 mM CaCl2 was added to the gel filtration buffer [50mM Tris/HCl/150mM NaCl (pH 8.0)], in lieu of an equimolar portion of NaCl. This minor compositional change induced significant differences in the venom elution profile on Superdex 200. For this reason, the influence of buffer composition on chromatographic behavior was investigated using an analytical Superdex 75 HR 10/30 column. Phospholipase (PLA) was used as a marker because Naja atra PLA had previously been observed to interact hydrophobically with this resin. PLA elution volumes generally increased as buffer pH decreased. Addition of 20% acetonitrile to the Tris buffer with CaCl2, reduced hydrophobic interaction of the PLA so significantly that its elution was non-overlapping in the two buffers. Other venom constituents, including bradykinin-potentiating peptides and probable hemorrhagic metalloproteases, were similarly affected. Buffer calcium, bound by vicinal dextran hydroxyl groups, appears to retard elution of this acidic PLA.
Asunto(s)
Bothrops/metabolismo , Cromatografía en Agarosa/métodos , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Resinas de Intercambio Iónico/química , Fosfolipasas/química , Fosfolipasas/aislamiento & purificación , Animales , Cromatografía por Intercambio Iónico/métodos , Venenos de Crotálidos/enzimología , Concentración de Iones de HidrógenoRESUMEN
Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).
Asunto(s)
Aspergillus/enzimología , Fraccionamiento Químico/métodos , Cromatografía de Afinidad/métodos , Cromatografía en Agarosa/métodos , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/aislamiento & purificación , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/aislamiento & purificación , Triazinas , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/aislamiento & purificación , Adsorción , Colorantes , Activación Enzimática , Glicósido Hidrolasas/química , Concentración de Iones de Hidrógeno , Complejos Multienzimáticos/química , Temperatura , beta-Glucosidasa/químicaRESUMEN
Rabbits are frequently used as models for studying coagulation and platelet disorders. However, few reports on literature have dealt with the purification and characterization of rabbit platelet proteins. Herein a protocol for the simultaneous purification of rabbit platelet factor 4 (PF4) and platelet glycoprotein IIb-IIIa (GPIIb-IIIa, integrin alpha(IIb)beta(3)) is described. Specific antibodies were raised in laying chicken, which were used for assaying PF4 by ELISA, and GPIIb-IIIa by direct immunofluorescence and flow cytometry. Furthermore, the binding of monoclonal antibodies specific for GPIIb-IIIa complex (P2), ligand-induced binding site of GPIIIa (LIBS1) and rabbit P-selectin (12A7), as well as of polyclonal IgY specific for rabbit GPIIb-IIIa, was compared in quiescent and thrombin-activated platelets. Polyclonal anti-rabbit PF4 IgY was a specific and sensitive probe that could be used for assaying PF4 in plasma samples. GPIIb-IIIa expression was increased in thrombin-activated platelets, as evaluated by flow cytometric analysis using P2 and polyclonal antibodies raised in chickens. Rabbit GPIIb-IIIa also exhibited a conformational modification that caused the appearance of ligand-induced binding sites. Increased P-selectin expression, used as a positive control, was also noticeable in thrombin-activated platelets. These data evidence that antibodies raised in laying chickens specific to rabbit PF4 and GPIIb-IIIa, as well as certain monoclonal antibodies specific for human GPIIb-IIIa, may be used for investigating rabbit platelet physiology.
Asunto(s)
Plaquetas/inmunología , Inmunoglobulinas/química , Factor Plaquetario 4/aislamiento & purificación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/aislamiento & purificación , Conejos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Pollos , Cromatografía en Agarosa , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Directa , Cobayas , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/inmunología , Masculino , Activación Plaquetaria/inmunología , Factor Plaquetario 4/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Trombina/inmunologíaRESUMEN
In the present work we propose a simple method for affinity purification of the 67-kDa lectin-like glycoprotein (LLGP-67) from Trypanosoma cruzi, the causative agent of Chagas' disease. The LLGP-67, which presents galactose binding activity and participates in the host cell recognition process, was previously purified by methods based on its interaction with galactose residues on erythrocytic membranes. We describe herein results showing that this protein can be purified from T. cruzi in a direct way using non-derivatized agarose as a chromatographic ligand. We also demonstrate the relevance of LLGP-67 as an antigen for human diagnosis of chagasic infection. Sensitivity and specificity for this antigen were calculated, being 98 and 98.11% respectively.
Asunto(s)
Antígenos de Protozoos/aislamiento & purificación , Enfermedad de Chagas/diagnóstico , Lectinas/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Trypanosoma cruzi/química , Animales , Antígenos de Protozoos/análisis , Antígenos de Protozoos/inmunología , Enfermedad de Chagas/parasitología , Cromatografía de Afinidad , Cromatografía en Agarosa , Ensayo de Inmunoadsorción Enzimática , Femenino , Galactosa/metabolismo , Humanos , Sueros Inmunes , Lectinas/análisis , Lectinas/inmunología , Proteínas Protozoarias/análisis , Proteínas Protozoarias/inmunología , Conejos , Trypanosoma cruzi/inmunologíaRESUMEN
A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose-Sepharose. The lectin content was about 265 mg/100g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5mM, and then p-nitrophenyl alpha-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two pI values of 5.8 and 6.1 and E(1%)(1 cm) of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS-PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca(2+) and Mn(2+) ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 degrees C for more than 90 min the lectin was fairly stable; however, when heated at 70 degrees C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 degrees C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.