RESUMEN

Kunitz proteinase inhibitors are widely distributed in legume seeds, and some of them have the ability to inhibit two different classes of enzymes. In this report, novel insights into three-dimensional structure and action mechanism of ApKTI, an Adenanthera pavonina Kunitz trypsin inhibitor, were provided to shed some light on an unconventional non-competitive activity against trypsin and papain. Firstly, ApKTI was purified by two tandem-size molecular exclusion chromatography high resolutions, Sephacryl S-100 and Superose 12 10/300 GL. Purified ApTKI showed molecular mass of 22 kDa and higher affinity against trypsin in comparison to papain, while the bifunctional inhibitor presented lower inhibitory activity. Moreover, in vitro assays showed that ApKTI has two independent interaction sites, permitting simultaneous inhibition to both enzymes. Theoretical three-dimensional structures of ApTKI complexed to both target proteinases were constructed in order to determine interaction mode by using Modeller v9.6. Since the structure of no non-competitive Kunitz inhibitor has been elucidated, ApTKI-trypsin and ApTKI-papain docking were carried out using Hex v5.1. In silico experiments showed that the opposite inhibitor loop interacts with adjacent sites of trypsin (Arg(64), Ser(107), Arg(88) and Lys(108)) and papain (Gln(51), Asp(172) and Arg(173)), probably forming a ternary complex. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. The predicted non-coincidence of trypsin and papain binding sites is completely different from that of previously proposed inhibitors, adding more information about mechanisms of non-competitive plant proteinase inhibitors.

Asunto(s)

Proteasas de Cisteína/metabolismo , Fabaceae/química , Modelos Moleculares , Péptidos/química , Péptidos/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Semillas/química , Serina Proteasas/metabolismo , Secuencia de Aminoácidos , Proteasas de Cisteína/química , Datos de Secuencia Molecular , Papaína/química , Papaína/metabolismo , Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Serina Proteasas/química , Volumetría , Tripsina/química , Tripsina/metabolismo

RESUMEN

Seeds from legumes including the Gilcine max are known to be a rich source of protease inhibitors. The soybean Kunitz trypsin inhibitors (SKTIs) have been well characterised and have been found to exhibit many biological activities. However their effects on inflammatory diseases have not been studied to date. In this study, SKTI was purified using anion exchange chromatography using a Resource Q column. The purified protein was able to inhibit human neutrophil elastase (HNE) and bovine trypsin. Purified SKTI inhibited HNE with an IC(50) value of 8mug or 0.3nM. At this concentration SKTI showed neither cytotoxic nor haemolytic effects on human blood cell populations. SKTI showed no deleterious effects on organs, blood cells or the hepatic enzymes ALT and AST in the mouse model of acute systemic toxicity. Human neutrophils incubated with SKTI released less HNE than control neutrophils when stimulated with PAF or fMLP (83.1% and 70% respectively). These results showed that SKTI affected both pathways of elastase release by PAF and fMLP stimuli, suggesting that SKTI is an antagonist of fMLP/PAF receptors. In an in vivo mouse model of LPS acute lung injury, SKTI significantly suppressed the inflammatory effects caused by elastase in a dose-dependent manner. Histological sections stained by hematoxylin/eosin confirmed this decrease in inflammation. These results showed that SKTI could be used as a pharmacological agent for the therapy of many inflammatory diseases.

Asunto(s)

Lesión Pulmonar Aguda/tratamiento farmacológico , Antiinflamatorios/farmacología , Elastasa de Leucocito/efectos de los fármacos , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Lesión Pulmonar Aguda/fisiopatología , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/toxicidad , Bovinos , Cromatografía por Intercambio Iónico/métodos , Modelos Animales de Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Concentración 50 Inhibidora , Elastasa de Leucocito/metabolismo , Masculino , Ratones , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Receptores de Formil Péptido/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Semillas , Glycine max/química , Pruebas de Toxicidad Aguda , Tripsina/efectos de los fármacos , Tripsina/metabolismo , Inhibidor de la Tripsina de Soja de Kunitz/administración & dosificación , Inhibidor de la Tripsina de Soja de Kunitz/toxicidad

RESUMEN

A novel pathogenesis-related class 10 (PR-10) protein with papain inhibitory activity, named CpPRI, was purified from Crotalaria pallida roots by ammonium sulfate precipitation followed by three reverse-phase high-performance liquid chromatographies (HPLCs). CpPRI is made up of a single polypeptide chain with a M(r) of 15 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein exhibited a K(i) value of 1.8 x 10(-9) M and operates via a noncompetitive inhibition mechanism. The alignment of the N-terminal amino acid sequence of CpPRI with other proteins revealed its identity with PR-10 proteins. CpPRI acts against digestive proteinase from root-knot nematode Meloidogyne incognita and demonstrated nematostatic and nematicide effects on this parasite in bioassays. In a localization study, fluorescein-5-isothiocyanate (FITC)-CpPRI was observed to internalize and diffuse over the entire J2 body after 6 h of incubation. This fact could explain the natural tolerance of this plant species to nematodes.

Asunto(s)

Crotalaria/química , Inhibidores Enzimáticos/farmacología , Papaína/antagonistas & inhibidores , Enfermedades de las Plantas/parasitología , Proteínas de Plantas/farmacología , Tylenchoidea/efectos de los fármacos , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Cinética , Solanum lycopersicum/parasitología , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas/parasitología , Tylenchoidea/fisiología

RESUMEN

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 degrees C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.

Asunto(s)

Hemaglutinación/efectos de los fármacos , Lactosa/metabolismo , Lectinas/aislamiento & purificación , Lectinas/farmacología , Leishmania/efectos de los fármacos , Leishmania/inmunología , Poríferos/química , Animales , Bovinos , Humanos , Lectinas/química , Lectinas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad por Sustrato

RESUMEN

An actual severe problem in agriculture consists of an expressive increase of economical losses caused by fungi and resistant bacteria toward antibiotics. In order to find a solution to this problem, several studies have been concentrating on the screening of novel plant defense peptides with antimicrobial activities. These peptides are commonly characterized by having low molecular masses and cationic charges. The present work reports the purification and characterization of a novel plant peptide with molecular mass of 5340 Da, named Cp-AMP, from seeds of C. pallida, a typical plant from Caatinga biome. Purification was achieved using a size exclusion S-200 column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Cp-AMP was able to inhibit the development of filamentous fungi Fusarium oxysporum as well as the gram-negative bacterium Proteus sp. The identification of Cp-AMP could contribute, in the near future, to the development of biotechnological products, such as transgenic plants with enhanced resistance to pathogenic fungi and/or of antibiotics production derived from plant sources in order to control bacterial infections.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Crotalaria/química , Fusarium/efectos de los fármacos , Proteus/efectos de los fármacos , Semillas/química , Péptidos Catiónicos Antimicrobianos/química , Cromatografía Liquida/métodos , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología

RESUMEN

PURPOSE: In this study, the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians was studied in different cancer cell lines. METHODS: CvL cytotoxicity was evaluated in mammalian tumor cells and in normal human peripheral blood lymphocytes by the MTT assay using the same range of concentrations (1-150 microg ml(-1)). The mechanisms involved in K562 cell death were investigated by confocal fluorescence microscopy, flow cytometry and immunoblot. RESULTS: CvL inhibited the growth of human leukemia cells, with IC(50) values of 70 and 100 microg ml(-1) for K562 and JURKAT cells, respectively, but it was ineffective on blood lymphocytes and solid tumor cell lines. K562 cell death occurred 72 h after exposure to the lectin and with signs of apoptosis, as analyzed by DAPI and annexin V/PI staining. Investigation of the possible mediators of this process showed that cell death occurred via a caspase-independent pathway. Confocal fluorescence microscopy indicated a pivotal role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished CvL cytotoxic effect. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFkappaB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and reduced expression of pRb, suggesting that CvL can induce cell cycle arrest. CONCLUSIONS: Collectively, these findings indicate an antileukemic effect for CvL and suggest that cathepsin B acts as a death mediator in CvL-induced cytotoxicity possibly in an uncharacterized connection with the membrane death receptor pathway.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Clione/química , Lectinas/farmacología , Animales , Antineoplásicos/aislamiento & purificación , Caspasas/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Immunoblotting , Concentración 50 Inhibidora , Células Jurkat , Células K562 , Lectinas/aislamiento & purificación , Linfocitos/citología , Linfocitos/efectos de los fármacos

RESUMEN

The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.

Asunto(s)

Insecticidas , Lepidópteros/enzimología , Lepidópteros/crecimiento & desarrollo , Péptido Hidrolasas/metabolismo , Proteínas de Plantas/farmacología , Inhibidores de Tripsina/farmacología , Animales , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Tracto Gastrointestinal/enzimología , Concentración de Iones de Hidrógeno , Larva/enzimología , Larva/crecimiento & desarrollo , Proteínas de Almacenamiento de Semillas , Glycine max/química

RESUMEN

Erythrina velutina vicilin, EvV, is a dimeric glycoprotein with Mr of 124.6 kDa. EvV was tested for anti-insect activity against bean bruchid larvae. EvV had LD(50) of 0.10% and ED(50) of 0.14% for Z. subfasciatus and LD(50) of 0.26% and ED(50) of 0.19% for C. maculatus. EvV was not digested by bean larvae enzymes until 12 h of incubation, and at 24 h EvV was more resistant to Z. subfasciatus enzymes.

Asunto(s)

Quitina/metabolismo , Escarabajos/efectos de los fármacos , Erythrina/química , Proteínas de Plantas/farmacología , Animales , Escarabajos/clasificación , Escarabajos/crecimiento & desarrollo , Larva/efectos de los fármacos , Control Biológico de Vectores , Proteínas de Plantas/metabolismo , Proteínas de Almacenamiento de Semillas , Semillas/química

RESUMEN

Chitin-binding vicilin from Erythrina velutina seeds was purified by ammonium sulfate followed by affinity chromatography on a chitin column and gel filtration on Superose-6-10-300-GL. The Erythrina velutina vicilin, called EvV, is a tetrameric glycoprotein composed of 1.85% carbohydrates and M r of 216.6 kDa, consisting of two subunits of M r of 54.8 and two subunits of M r of 50.8 kDa. The EvV homogeneity was confirmed in native PAGE where it was observed to be a unique acid-protein band with slow mobility in this gel. Effect of EvV on C. capitata larvae was examined by bioassay and its mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV when added to diet caused strong effect on mortality (ED50 of 0.14%) and larval mass (WD50 of 0.12%). These deleterious effects were associated to the binding to chitin structures present in peritrophic membrane and to gut epithelial cells, and its low digestibility in C. capitata digestive tract. These results are the first demonstration of a proteinaceous bioinsecticide from plant origin effective against C. capitata larvae. EvV may be part of the pest management programs or an alternative in plant improvement program.

Asunto(s)

Ceratitis capitata/crecimiento & desarrollo , Quitina/metabolismo , Erythrina/química , Insecticidas/química , Larva/crecimiento & desarrollo , Proteínas de Plantas/aislamiento & purificación , Animales , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Almacenamiento de Semillas , Semillas/química

RESUMEN

CvL, a lectin from the marine sponge Cliona varians agglutinated type A papainized erythrocytes and was strongly inhibited by d-galactose and sucrose. Models of leukocyte migration in vivo were used to study the inflammatory activity of CvL through of mouse paw oedema and peritonitis. Effect of CvL on peritoneal macrophage activation was analysed. Effects of corticoids and NSAIDS drugs were also evaluated on peritonitis stimulated by CvL. Results showed that mouse hind-paw oedema induced by subplantar injections of CvL was dose dependent until 50 microg/cavity. This CvL dose when administered into mouse peritoneal cavities induced maxima cell migration (9283 cells/microL) at 24 h after injection. This effect was preferentially inhibited by incubation of CvL with the carbohydrates d-galactose followed by sucrose. Pre-treatment of mice with 3% thioglycolate increases the peritoneal macrophage population 2.3 times, and enhanced the neutrophil migration after 24 h CvL injection (75.8%, p<0.001) and no significant effect was observed in the presence of fMLP. Finally, pre-treatment of mice with dexamethasone (cytokine antagonist) decreased (65.6%, p<0.001), diclofenac (non-selective NSAID) decreased (34.5%, p<0.001) and Celecoxib (selective NSAID) had no effect on leukocyte migration after submission at peritonitis stimulated by CvL, respectively. Summarizing, data suggest that CvL shows pro-inflammatory activity, inducing neutrophil migration probably by pathway on resident macrophage activation and on chemotaxis mediated by cytokines.

Asunto(s)

Quimiotaxis de Leucocito/efectos de los fármacos , Edema/inducido químicamente , Inflamación/inducido químicamente , Lectinas/farmacología , Peritonitis/inducido químicamente , Poríferos/química , Animales , Antiinflamatorios no Esteroideos/farmacología , Celecoxib , Dexametasona/farmacología , Diclofenaco/farmacología , Modelos Animales de Enfermedad , Edema/fisiopatología , Inflamación/fisiopatología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos , Peritonitis/fisiopatología , Pirazoles/farmacología , Sulfonamidas/farmacología

RESUMEN

A novel trypsin-papain inhibitor, named PdKI-2, was purified from the seeds of Pithecelobium dumosum seeds by TCA precipitation, Trypsin-Sepharose chromatography and reversed-phase HPLC. PdKI-2 had an M(r) of 18.1 kDa as determined by SDS-PAGE and was composed of a single polypeptide chain. The inhibition on trypsin was stable at pH range 2-10, temperature of 50 degrees C and had a K(i) value of 1.65 x 10(-8)M, with a competitive inhibition mechanism. PdKI-2 was also active to papain, a cysteine proteinase, and showed a noncompetitive inhibition mechanism and K(i) value of 5.1 x 10(-7)M. PdKI-2 was effective against digestive proteinase from bruchids Zabrotes subfasciatus and Callosobruchus maculatus; Dipteran Ceratitis capitata; Lepidopterans Plodia interpunctella and Alabama argillacea, with 74.5%, 70.0%, 70.3%, 48.7%, and 13.6% inhibition, respectively. Results support that PdKI-2 is a member of Kunitz-inhibitor family and its effect on digestive enzyme larvae from diverse orders indicated this protein as a potent insect antifeedant.

Asunto(s)

Sistema Digestivo/enzimología , Papaína/antagonistas & inhibidores , Inhibidores de Proteasas/aislamiento & purificación , Semillas/metabolismo , Inhibidores de Tripsina/aislamiento & purificación , Animales , Dípteros/enzimología , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/metabolismo , Insectos/enzimología , Cinética , Lepidópteros/enzimología , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Temperatura , Inhibidores de Tripsina/metabolismo , Inhibidores de Tripsina/farmacología

RESUMEN

Chitin-binding vicilin from Enterolobium contortisiliquum seeds was purified by ammonium sulfate followed by gel filtration on Sephacryl 300-SH and on Sephacryl 200-SH. The vicilin, called EcV, is a dimeric glycoprotein composed of 1.03% carbohydrates and a Mr of 151 kDa, consisting of two subunits of Mr of 66.2 and 63.8 kDa. The EcV homogeneity was confirmed in a PAGE where it was observed to be a unique acid protein band with slow mobility in this native gel. E. contortisiliquum vicilin (EcV) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae and for phytopathogenic fungi, F. solani and C. lindemuntianum. EcV was very effective against both bruchids, producing 50% mortality for Z. subfasciatus at an LD50 of 0.43% and affected 50% of the larvae mass with an ED50 of 0.65%. In artificial diets given to C. maculatus, 50% of the larvae mass was affected with an ED50 of 1.03%, and larva mortality was 50% at LD50 of 1.11%. EcV was not digested by midgut homogenates of C. maculatus and Z. Subfasciatus until 12 h of incubation, and at 24 h EcV was more resistant to Z. subfasciatus larval proteases. The binding to chitin present in larvae gut associated to low EcV digestibility could explain its lethal effects. EcV also exerted an inhibitory effect on the germination of F. solani at concentrations of 10 and 20 microg mL-1. The effect of EcV on fungi is possibly due to binding to chitin-containing structures of the fungal cell wall.

Asunto(s)

Quitina/metabolismo , Fabaceae/química , Fungicidas Industriales/farmacología , Insecticidas , Proteínas de Plantas/farmacología , Semillas/química , Animales , Escarabajos , Fungicidas Industriales/aislamiento & purificación , Insecticidas/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Proteínas de Almacenamiento de Semillas

RESUMEN

CvL, a lectin from the marine sponge Cliona varians was purified by acetone fractionation followed by Sepharose CL 4B affinity chromatography. CvL agglutinated papainized treated human erythrocytes with preference for type A erythrocytes. The lectin was strongly inhibited by monosaccharide d-galactose and disaccharide sucrose. CvL is a tetrameric glycoprotein of 28 kDa subunits linked by disulphide bridges with a molecular mass of 106 kDa by SDS-PAGE and 114 kDa by Sephacryl S300 gel filtration. The lectin was Ca2+ dependent, stable up to 60 degrees C for 60 min, with optimum pH of 7.5. CvL displays a cytotoxic effect on gram positive bacteria, such as Bacillus subtilis and Staphylococcus aureus. However, CvL did not affect gram negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Leishmania chagasi promastigotes were agglutinated by CvL up to 2(8) titer. These findings are indicative of the physiological defense roles of CvL and its possible use in the antibiosis of bacteria and protozoa pathogenic.

Asunto(s)

Antibacterianos/farmacología , Antiprotozoarios/farmacología , Lectinas/aislamiento & purificación , Lectinas/farmacología , Poríferos/química , Animales , Bacillus subtilis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pruebas de Hemaglutinación , Humanos , Lectinas/química , Leishmania infantum/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos

RESUMEN

The digestive system of Ceratitis capitata was characterized during its larval development and in the insect stage. Disaccharidases against maltose and sucrose were more evident in the 2nd and 3rd day of larval development and in the adult stage, respectively. Glycosil-hydrolyses with higher specific alpha-galactosidasic and beta-galactosidasic activities were detected in the 2nd and 3rd day of the larval stage, respectively. Specific proteolytic activities against azocasein showed an increase in the 4th and 5th day of the larval stage and in the adult stage. Specific hemoglobin activities were constant between 2nd and 6th day of the larval stage. The larvae used mainly serine proteinases, such as trypsin/chymotrypsin, and the adult insects only chymotrypsin-like enzymes in their digestive process. Two serine proteinases were separated from zymogram between the 4th and 5th day of larval development and in the adult stage. Effect of soybean trypsin inhibitor (SBTI, a serine proteinase inhibitor) on development of C. capitata was examined by bioassay. C. capitata was susceptible to SBTI which affected larval mass at ED50 3.01%.

Asunto(s)

Dípteros/enzimología , Serina Endopeptidasas/metabolismo , Inhibidor de la Tripsina de Soja de Kunitz/farmacología , Animales , Caseínas/farmacología , Dípteros/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas/metabolismo

RESUMEN

A proteinaceous inhibitor with high activity against trypsin-like serine proteinases was purified from seeds of the tamarind tree (Tamarindus indica) by gel filtration on Shephacryl S-200 followed by a reverse-phase HPLC Vidac C18 TP. The inhibitor, called the tamarind trypsin inhibitor (TTI), showed a Mr of 21.42 kDa by mass spectrometry analysis. TTI was a noncompetitive inhibitor with a Ki value of 1.7 x 10(-9) M. In vitro bioinsecticidal activity against insect digestive enzymes from different orders showed that TTI had remarkable activity against enzymes from coleopteran, Anthonomus grandis (29.6%), Zabrotes subfasciatus (51.6%), Callosobruchus maculatus (86.7%), Rhyzopertha dominica(88.2%), and lepidopteron, Plodia interpuncptella (26.7%), Alabama argillacea (53.8%), and Spodoptera frugiperda (75.5%). Also, digestive enzymes from Diptera, Ceratitis capitata (fruit fly), were inhibited (52.9%). In vivo bioinsecticidal assays toward C. capitata and C. maculatus larvae were developed. The concentration of TTI (w/w) in the artificial seed necessary to cause 50% mortality (LD50) of larvae was 3.6%, and that to reduce mass larvae by 50.0% (ED50) was 3.2%. Furthermore, the mass C. capitata larvae were affected at 53.2% and produced approximately 34% mortality at a level of 4.0% (w/w) of TTI incorporated in artificial diets.

Asunto(s)

Ceratitis capitata , Insecticidas , Semillas/química , Tamarindus/química , Inhibidores de Tripsina , Gorgojos , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Inhibidores de Tripsina/aislamiento & purificación

RESUMEN

A beta-glucuronidase was purified from Pomacea sp. eggs by ammonium sulfate fractionation, DEAE-BioGel and Heparin-Sepharose chromatographies. This enzyme showed a Mr 180 kDa, with subunits of 90 kDa. The kinetic parameters were: pH 4.0, temperature 60 degrees C, Km 2.7 x 10(-6) and Vmax 15.3 microM/h, activator Mg+2, and inhibitor: lactone of D-saccharic acid. beta-glucuronidase is an exoglucuronidase involved in glycosaminoglycans metabolism with kinetics parameters similar to those found in mammals.

Asunto(s)

Glucuronidasa/aislamiento & purificación , Glucuronidasa/metabolismo , Moluscos/embriología , Moluscos/enzimología , Animales , Desarrollo Embrionario , Estabilidad de Enzimas , Glucuronidasa/química , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Moluscos/clasificación , Temperatura

RESUMEN

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.

Asunto(s)

Ceratitis capitata/enzimología , Crotalaria/química , Insecticidas , Inhibidores de Tripsina , Gorgojos/enzimología , Animales , Ceratitis capitata/crecimiento & desarrollo , Quimotripsina/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Insecticidas/aislamiento & purificación , Larva/enzimología , Dosificación Letal Mediana , Elastasa Pancreática/antagonistas & inhibidores , Semillas/metabolismo , Temperatura , Inhibidores de Tripsina/aislamiento & purificación , Gorgojos/crecimiento & desarrollo

RESUMEN

Alpha-amylase Inhibitors were isolated from Ficus sp. (Gameleira) seeds by acetone fractionation and Sephadex G-50. Two inhibitors (alpha-PPAI and alpha-ZSAI) were tested against alpha-amylases from coleopteran larvae. alpha-PPAI was active to alpha-amylases of Callosobruchus maculatus (52%) and Zabrotes subfasciatus (53%). alpha-ZSAI was strongly active to Z. subfasciatus (100%) of and Mimosestes mimosae (98%). The alpha-ZSAI is a glycoprotein of approximately 50 kDa with an IC50 value of 0.074 microg microl(-1).

Asunto(s)

Escarabajos/efectos de los fármacos , Ficus/química , Control de Plagas , Proteínas de Plantas/química , Animales , Escarabajos/enzimología , Larva/efectos de los fármacos , Larva/enzimología , Proteínas de Plantas/toxicidad , Semillas/química , Inhibidores de Tripsina , alfa-Amilasas/antagonistas & inhibidores

RESUMEN

We have confirmed here that the seeds of the common bean (Phaseolus vulgaris, L.) do not support development of the bruchid Callosobruchus maculatus (F.), a pest of cowpea [Vigna unguiculata (L.) Walp] seeds. Analysis of the testa (seed coat) of the bean suggested that neither thickness nor the levels of compounds such as tannic acid, tannins, or HCN are important for the resistance. On the other hand, we have found that phaseolin (vicilin-like 7S storage globulin), detected in the testa by Western blotting and N-terminal amino acid sequencing, is detrimental to the development of C. maculatus. As for the case of other previously studied legume seeds (Canavalia ensiformis and Phaseolus lunatus) we suggest that the presence of vicilin-like proteins in the testa of P. vulgaris may have had a significant role in the evolutionary adaptation of bruchids to the seeds of leguminous plants.

Asunto(s)

Escarabajos/efectos de los fármacos , Productos Agrícolas/parasitología , Phaseolus/química , Proteínas de Plantas/farmacología , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Phaseolus/parasitología , Proteínas de Plantas/aislamiento & purificación , Proteínas de Almacenamiento de Semillas , Semillas/química , Semillas/parasitología

RESUMEN

We have confirmed here that the seeds of the common bean (Phaseolus vulgaris, L.) do not support development of the bruchid Callosobruchus maculatus (F.), a pest of cowpea [Vigna unguiculata (L.) Walp] seeds. Analysis of the testa (seed coat) of the bean suggested that neither thickness nor the levels of compounds such as tannic acid, tannins, or HCN are important for the resistance. On the other hand, we have found that phaseolin (vicilin-like 7S storage globulin), detected in the testa by Western blotting and N-terminal amino acid sequencing, is detrimental to the development of C. maculatus. As for the case of other previously studied legume seeds (Canavalia ensiformis and Phaseolus lunatus) we suggest that the presence of vicilin-like proteins in the testa of P. vulgaris may have had a significant role in the evolutionary adaptation of bruchids to the seeds of leguminous plants.

Asunto(s)

Animales , Femenino , Escarabajos , Phaseolus nanus , Western Blotting