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1.
Int J Biol Macromol ; 164: 3340-3348, 2020 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-32871119

RESUMEN

The bioactive form of thermostable and alkali stable pectinase of Bacillus pumilus dcsr1 is a homodimer of the molecular mass of 60 kDa with a pI of 4.6. The enzyme is optimally active at 50 °C and pH 10.5, and its Michaelis constant (Km), maximum rate of reaction (Vmax), activation energy (Ea), and temperature quotient (Q10) values (for citrus pectin) are 0.29 mg mL-1, 116 µmole mg-1 min-1, 74.73 KJmol-1 and 1.57, respectively. The enzyme has a shelf life of one and a half years at room temperature as well as 4 °C. The activity of the enzyme is stimulated by Mn2+ and Ca2+ and inhibited by Hg+, Cd2+, Co2+, Zn2+, Fe2+, Pb2+, EDTA and urea to a varied extent. The conformational studies of the enzyme revealed a high ß-sheet content in the bioactive dimer, and high α-helix in the inactive monomer. The Circular Dichroism (CD) spectra of the dimer in the presence of inhibitors suggested a marked decrease in ß-sheet, and a significant increase in α-helix, suggesting a key role of ß-sheets in the enzyme catalysis. Based on the end product analysis, the enzyme is an exopolygalacturonase with a unique ability of transglycosylation. When ramie fibers were treated with the enzyme, removal of gummy material (pectin) was visible, confirming its applicability in the degumming process.


Asunto(s)
Bacillus pumilus/enzimología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Bacillus/enzimología , Bacillus pumilus/metabolismo , Proteínas Bacterianas/química , Boehmeria/química , Boehmeria/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Pectinas/química , Poligalacturonasa/química , Polisacárido Liasas/química , Especificidad por Sustrato , Temperatura
2.
Methods Mol Biol ; 2020: 185-205, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31177501

RESUMEN

House dust mites are globally significant triggers of allergic disease. Notable among their extensive repertoire of allergens are the Group 1 cysteine peptidase allergens which function as digestive enzymes in house dust mites. Compelling evidence suggests that the proteolytic activity of these molecules plays a key role in the development and maintenance of allergic diseases through the activation of innate immune mechanisms which exploit genetic predispositions to allergy. Growing interest in this area creates a requirement for high-quality purified protein, whether natural or recombinantly expressed. It has also identified these allergens as therapeutic targets for a novel approach to allergy treatment through modulation of innate immune responses. The purpose of this chapter is to describe a new method for the purification of Der p 1 and use of the protein produced in a screening assay designed for the discovery of novel inhibitors of Group 1 house dust mite allergens.


Asunto(s)
Antígenos Dermatofagoides/química , Antígenos Dermatofagoides/aislamiento & purificación , Proteínas de Artrópodos/química , Proteínas de Artrópodos/aislamiento & purificación , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Pyroglyphidae/inmunología , Animales , Proteínas de Artrópodos/antagonistas & inhibidores , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Estructura Molecular , Proteolisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad
3.
J Microbiol Biotechnol ; 28(9): 1426-1432, 2018 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-30369109

RESUMEN

Staphylococcus aureus (S. aureus) causes a broad variety of diseases. The spread of multidrugresistant S. aureus highlights the need to develop new ways to combat S. aureus infections. Sortase A (SrtA) can anchor proteins containing LPXTG binding motifs to the bacteria surface and plays a key role in S. aureus infections, making it a promising antivirulence target. In the present study, we used aSrtA activity inhibition assay to discover that isovitexin, a Chinese herbal product, can inhibit SrtA activity with an IC50 of 28.98 µg/ml. Using a fibrinogenbinding assay and a biofilm formation assay, we indirectly proved the SrtA inhibitory activity of isovitexin. Additionally, isovitexin treatment decreased the amount of staphylococcal protein A (SpA) on the surface of the cells. These data suggest that isovitexin has the potential to be an anti-infective drug against S. aureus via the inhibition of sortase activity.


Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Antiinfecciosos/farmacología , Apigenina/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Biopelículas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Aminoaciltransferasas/genética , Aminoaciltransferasas/aislamiento & purificación , Aminoaciltransferasas/metabolismo , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Medicamentos Herbarios Chinos/farmacología , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Staphylococcus aureus/enzimología , Staphylococcus aureus/patogenicidad , Factores de Virulencia/metabolismo
4.
Asian Pac J Allergy Immunol ; 33(1): 42-51, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25840633

RESUMEN

BACKGROUND: House dust mite (HDM) induced matrix metalloproteinase (MMP)-9 plays a role in asthma. Zingiber cassumunar Roxb. (Phlai in Thai) has been used in folk medicine for asthma treatment. OBJECTIVE: We investigated effects of Phlai and its constituent (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (compound D) on the cleavage of pro- MMP-9 by HDM. The effects of these compounds on phorbol 12-myristate 13-acetate (PMA)- induced MMP-9 gene and protein expression in airway epithelial cells (NCI-H292) were also investigated. METHODS: Pro-MMP-9 was directly activated in vitro with HDM in the presence or absence of the ethanolic extracts of Phlai or compound D for 1 hour. The amount of activated MMP-9 was determined using gelatin zymography. To study the cellular response of Phlai, NCI-H292 cells were pretreated with crude Phlai extracts or compound D for 2 hours, and then the cells were stimulated with PMA for 48 hours. The mRNA RT-PCR and Western blotting, respectively. MMP-9 activity was determined by gelatin zymography. RESULTS: Crude Phlai extracts (0.25 - 2.0 mg/ml) and compound D (0.5 - 4.0 mg/ml) inhibited pro- MMP-9 cleavage by HDM. Furthermore, crude Phlai extracts (100 mg/ml) and compound D, at concentrations of 50 and 100 mg/ml, attenuated the PMA-induced MMP-9 gene and expression in NCI-H292 cells. These compound also suppressed MMP-9 release from PMA-induced NCI-H292 cells. CONCLUSION: The crude ethanolic extract of Z. cassumunar and its active constituent compound D inhibited the cleavage of pro-MMP-9 by HDM. They also inhibited PMA-induced MMP-9 gene and protein synthesis in human airway epithelial cells.


Asunto(s)
Antígenos Dermatofagoides/farmacología , Proteínas de Artrópodos/farmacología , Butanoles/farmacología , Cisteína Endopeptidasas/farmacología , Células Epiteliales/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Extractos Vegetales/farmacología , Pyroglyphidae/química , Zingiberaceae/química , Animales , Antígenos Dermatofagoides/aislamiento & purificación , Proteínas de Artrópodos/aislamiento & purificación , Butanoles/aislamiento & purificación , Línea Celular Tumoral , Cisteína Endopeptidasas/aislamiento & purificación , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Expresión Génica , Humanos , Metaloproteinasa 9 de la Matriz/genética , Ésteres del Forbol/farmacología , Preparaciones de Plantas/química , Mucosa Respiratoria/citología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo
5.
Protein J ; 33(2): 199-209, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24596120

RESUMEN

A 24,118 Da (MALDI-TOF) cysteine peptidase (EC 3.4.22.16) was purified from the latex extract of Cryptostegia grandiflora by two chromatographic steps involving ion exchange and Reverse-phase HPLC. The purified protein (Cg24-I) exhibits a single band profile following reduced SDS-PAGE and a unique amino terminal sequence, 1VPASIDWREKGTVL14, that is similar to other plant cysteine peptidases. Cg24-I displayed optimal proteolytic activity at pH 8.0 with 25 mM phosphate buffer. The proteolytic activity was inhibited with 0.2 mM E-64 and 1 mM iodoacetamide and was stimulated with 1 mM DTT. Cg24-I fully inhibited spore germination of phytopathogenic fungi Fusarium solani at a dose of 28.1 µg/mL. Its toxicity involves the membrane permeabilization of spores as probed by propidium iodide uptake. These results show that latex peptidases are part of the plant's defensive strategy against phytopathogens and that they most likely act synergistically with other recognized defensive proteins.


Asunto(s)
Antifúngicos/química , Apocynaceae/enzimología , Apocynaceae/microbiología , Cisteína Endopeptidasas/química , Extractos Vegetales/química , Secuencia de Aminoácidos , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Apocynaceae/química , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Fusariosis/microbiología , Fusariosis/prevención & control , Fusarium/efectos de los fármacos , Fusarium/crecimiento & desarrollo , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Proteolisis
6.
J Med Food ; 13(6): 1532-6, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20954802

RESUMEN

Chives have been used both as food and as medicine. Previously, two fibrinolytic enzymes, ATFE-I (90 kDa) and ATFE-II (55 kDa), were identified in chives (Allium tuberosum), a perennial herb. In the present work, ATFE-II was purified by ion-exchange chromatography followed by gel filtration. In addition, the enzyme properties of ATFE-I and ATFE-II were compared. The molecular mass and isoelectric point (pI value) of ATFE-II were 55 kDa and pI 4.0, respectively, as revealed using one- or two-dimensional fibrin zymography. ATFE-II was optimally active at pH 7.0 and 45°C. ATFE-II degraded the Aα-chain of human fibrinogen but did not hydrolyze the Bß-chain or the γ-chain, indicating that the enzyme is an α-fibrinogenase. The proteolytic activity of ATFE-II was completely inhibited by 1 mM leupeptin, indicating that the enzyme belongs to the cysteine protease class. ATFE-II was also inhibited by 1 mM Fe²(+). ATFE-II exhibited high specificity for MeO-Suc-Arg-Pro-Tyr-p-nitroaniline (S-2586), a synthetic chromogenic substrate of chymotrypsin. Thus proteolytic enzymes from A. tuberosum may be useful as thrombolytic agents.


Asunto(s)
Cebollino/enzimología , Cisteína Endopeptidasas , Descubrimiento de Drogas , Fibrinólisis , Fibrinolíticos , Componentes Aéreos de las Plantas/enzimología , Proteínas de Plantas , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Compuestos Ferrosos/farmacología , Fibrinógeno/metabolismo , Fibrinolíticos/química , Fibrinolíticos/aislamiento & purificación , Fibrinolíticos/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Punto Isoeléctrico , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Leupeptinas/farmacología , Peso Molecular , Oligopéptidos/metabolismo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Especificidad por Sustrato , Temperatura , Terapia Trombolítica , Trombosis/tratamiento farmacológico
7.
J Biochem ; 147(5): 735-41, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20139060

RESUMEN

Ion-exchange chromatography (IEC) is the most frequently used chromatographic technique for the separation of proteins and peptides. In this article, the effects of urea on IEC separation of kiwifruit actinidin, egg white and urinary proteins were examined. The purity and relative amount of each protein in different conditions (in the presence or absence of urea) were compared with each other. The three parameters, including resolution, selectivity and efficiency of column in the presence of urea, were calculated and compared with the absence of urea. The results revealed that urea improved the purity of proteins and the resolution, selectivity and efficiency of IEC in separation of studied proteins.


Asunto(s)
Proteínas/química , Proteínas/aislamiento & purificación , Urea/química , Animales , Pollos , Cromatografía por Intercambio Iónico , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Clara de Huevo/química , Femenino , Frutas/enzimología , Humanos , Extractos Vegetales/química , Embarazo
8.
Int J Biol Macromol ; 43(3): 238-44, 2008 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-18590760

RESUMEN

Reported here is the overexpression, purification and partial characterization of recombinant coxsakievirus B3 2A protease (CVB3 2Apro) from bacterial cells transformed with a plasmid containing the CVB3 2Apro cDNA sequences. The structural investigation showed that the protein contains mostly beta-strand elements and requires Zn2+ ions as a structural component which appeared to be inhibitory if added exogenously. The purified enzyme activity was optimal at 4 degrees C and had a short half-life at physiological temperature. This feature can be the result of the presence of a high content of beta-structure and also hydrophobic residues in its structure.


Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Enterovirus Humano B/enzimología , Escherichia coli/genética , Expresión Génica , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Dicroismo Circular , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Evaluación Preclínica de Medicamentos , Enterovirus Humano B/genética , Inhibidores Enzimáticos/farmacología , Humanos , Datos de Secuencia Molecular , Desnaturalización Proteica , Espectrometría de Fluorescencia , Temperatura , Rayos Ultravioleta , Proteínas Virales/química , Proteínas Virales/genética , Zinc/farmacología
9.
Protein J ; 27(5): 267-75, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18478320

RESUMEN

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.


Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Frutas/enzimología , Frutas/crecimiento & desarrollo , Solanaceae/enzimología , Solanaceae/crecimiento & desarrollo , Secuencia de Aminoácidos , Dominio Catalítico , Resinas de Intercambio de Catión , Cisteína Endopeptidasas/química , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Extractos Vegetales/metabolismo
10.
J Neurosci ; 28(17): 4331-5, 2008 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-18434511

RESUMEN

Cowhage spicules provide an important model for histamine-independent itch. We determined that the active component of cowhage, termed mucunain, is a novel cysteine protease. We isolated mucunain and demonstrate that both native and recombinant mucunain evoke the same quality of itch in humans. We also show that mucunain is a ligand for protease-activated receptors two and four. These results support and expand the relationship between proteases, protease-activated receptors, and itch.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Mucuna/enzimología , Prurito/enzimología , Receptores Proteinasa-Activados/metabolismo , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/toxicidad , Células HeLa , Humanos , Ligandos , Mucuna/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Extractos Vegetales/toxicidad , Estructuras de las Plantas/enzimología , Estructuras de las Plantas/toxicidad , Prurito/inducido químicamente
11.
Plant Physiol Biochem ; 46(4): 403-13, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18207414

RESUMEN

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.


Asunto(s)
Cisteína Endopeptidasas/química , Ajo/enzimología , Hemaglutininas/química , Proteínas de Plantas/química , Animales , Caseínas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/aislamiento & purificación , Eritrocitos/química , Ajo/química , Ajo/genética , Gelatina/química , Pruebas de Hemaglutinación , Hemaglutininas/genética , Hemaglutininas/aislamiento & purificación , Hemoglobinas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Conejos , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
12.
Protein J ; 27(2): 88-96, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17932734

RESUMEN

Bromelia pinguin L. is a plant broadly distributed in Central America and Caribbean islands. The fruits have been used in traditional medicine as anthelmintic, probably owed to the presence of a mixture of cysteine endopeptidases, initially termed pinguinain. This work deals with the purification and characterization of the four main components of that mixture, two of them showing acid pI and the other two alkaline pI. Molecular masses (SDS-PAGE and MALDI-TOF), N-terminal sequence and the reactivity and kinetic parameters versus synthetic substrates (p-nitrophenyl-N-alpha-CBZ-amino acid esters, PFLNA, Z-Arg-Arg-p-NA, and Z-Phe-Arg-p-NA) of the studied peptidases are given, as well as the N-terminal sequences of the enzymes and the homology degree with other plant endopeptidases.


Asunto(s)
Bromelia/enzimología , Cisteína Endopeptidasas/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Cuba , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Frutas/enzimología , Focalización Isoeléctrica , Cinética , Datos de Secuencia Molecular , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato
13.
Phytomedicine ; 15(4): 237-44, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17689943

RESUMEN

Latex from Caricaceae contains proteolytic enzymes localized in the fruit, which are used ethnopharmacologically to treat digestive disorders. Some of these proteins display proliferative properties when probed with mammalian cells, suggesting a role in the reconstruction of wounded tissue. We tested the efficacy of a proteolytic fraction derived from Carica candamarcensis, designated as P1G10 in experimental rodent models, to protect and heal chemically induced gastric ulcers. The protective effect of oral administration of P1G10 fraction was analyzed in indomethacin-treated Wistar animals. The healing effect of P1G10 was studied following sub-serous injection of acetic acid in a Wistar rat model. The results show that P1G10 between 0.1 and 10 mg/kg protect indomethacin but not ethanol-induced gastric ulcers. The maximal protection attained was 67% with 10 mg/kg of P1G10. The healing rate by 10 mg/kg of P1G10 using the acetic acid ulcerogenic model is similar to that of omeprazole (10 mg/kg) or ranitidine (100 mg/kg). The effect of P1G10 at 10 mg/kg seems to be mediated by an increase in the mucus content by 25% and stimulation of angiogenesis by 64% in a manner similar to growth factors. These results confirm the protective and healing role of proteinases from C. candamarcensis.


Asunto(s)
Carica/enzimología , Cisteína Endopeptidasas/uso terapéutico , Látex/química , Fitoterapia , Úlcera Gástrica/tratamiento farmacológico , Animales , Antiulcerosos , Carica/química , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/farmacología , Frutas/química , Frutas/enzimología , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Ratas Wistar , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/prevención & control
14.
Biol Chem ; 387(8): 1063-74, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16895476

RESUMEN

SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms. We have synthesised and characterised the first generation of a red-shifted internally quenched fluorogenic substrate (RS-IQFS) for 3CLpro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 (Km and kcat values of 14 microM and 0.65 min-1). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CLpro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CLpro-mediated cleavage junctions of the SARS-CoV polyproteins. In contrast to BS-IQFSs, the RS-IQFS was not susceptible to fluorescence interference from coloured samples and allowed for successful screening of marine natural products and identification of a coumarin derivative, esculetin-4-carboxylic acid ethyl ester, a novel 3CLpro inhibitor (IC50=46 microM) and anti-SARS agent (EC50=112 microM; median toxic concentration>800 microM) from the tropical marine sponge Axinella corrugata.


Asunto(s)
Antivirales/química , Cisteína Endopeptidasas/química , Poríferos/química , Poríferos/clasificación , Inhibidores de Proteasas/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Espectrometría de Fluorescencia/métodos , Umbeliferonas/farmacología , Proteínas Virales/química , Animales , Antivirales/farmacología , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/aislamiento & purificación , Evaluación Preclínica de Medicamentos , Cinética , Estructura Molecular , Inhibidores de Proteasas/clasificación , Inhibidores de Proteasas/farmacología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Sensibilidad y Especificidad , Relación Estructura-Actividad , Factores de Tiempo , Umbeliferonas/química , Células Vero , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/aislamiento & purificación , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología
15.
J Ethnopharmacol ; 107(2): 189-98, 2006 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-16621376

RESUMEN

Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed.


Asunto(s)
Cisteína Endopeptidasas/farmacología , Plantas Medicinales , Esquistosomicidas/farmacología , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Malí , Péptidos/química , Péptidos/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Extractos Vegetales/farmacología , Plantas Medicinales/química , Plantas Medicinales/clasificación , Plantas Medicinales/enzimología , Esquistosomicidas/aislamiento & purificación , Especificidad por Sustrato
16.
Protein Pept Lett ; 13(1): 83-9, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16454675

RESUMEN

The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. The present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. The product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).


Asunto(s)
Bromelia/enzimología , Cisteína Endopeptidasas/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía por Intercambio Iónico , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/aislamiento & purificación , Cartilla de ADN , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
17.
Parasitology ; 132(Pt 5): 681-9, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16448585

RESUMEN

Extracts of plants, such as papaya, pineapple and fig, are known to be effective at killing intestinal nematodes that inhabit anterior sites in the small intestine, such as Heligmosomoides polygyrus. In this paper, we demonstrate that similar in vitro efficacy also occurs against a rodent nematode of the large intestine, Trichuris muris, and confirm that the cysteine proteinases present in the plant extracts are the active principles. The mechanism of action of these enzymes involved an attack on the structural proteins of the nematode cuticle, which was similar to that observed with H. polygyrus. However, not all plant cysteine proteinases were equally efficacious because actinidain, from the juice of kiwi fruit, had no detrimental effect on either the motility of the worms or the nematode cuticle. Papaya latex was also shown to significantly reduce both worm burden and egg output of mice infected with adult T. muris, demonstrating that enzyme activity survived passage to the caecum and was not completely inactivated by the acidity of the host's stomach or destroyed by the gastric or pancreatic proteinases. Thus, the cysteine proteinases from plants may be a much-needed alternative to currently available anthelmintic drugs due to their efficacy and novel mode of action against different gastrointestinal nematode species.


Asunto(s)
Antihelmínticos/farmacología , Cisteína Endopeptidasas/farmacología , Parasitosis Intestinales/parasitología , Fitoterapia , Tricuriasis/parasitología , Trichuris/efectos de los fármacos , Actinidia/química , Actinidia/enzimología , Ananas/química , Ananas/enzimología , Animales , Antihelmínticos/uso terapéutico , Carica/química , Carica/enzimología , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/uso terapéutico , Femenino , Ficus/química , Ficus/enzimología , Parasitosis Intestinales/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Movimiento/efectos de los fármacos , Recuento de Huevos de Parásitos , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Tricuriasis/tratamiento farmacológico , Trichuris/ultraestructura
18.
J Insect Physiol ; 52(1): 21-8, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16243350

RESUMEN

A unique 33-kDa cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM in vitro, dissected PMs were treated with purified, recombinant Mir1-CP and the movement of Blue Dextran 2000 across the PM was measured. Mir1-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and more actively permeabilized the PM than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrinia nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro and is critical to insect defense in maize.


Asunto(s)
Cisteína Endopeptidasas/farmacología , Spodoptera/efectos de los fármacos , Zea mays/enzimología , Animales , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Sistema Digestivo/efectos de los fármacos , Hemolinfa/química , Concentración de Iones de Hidrógeno , Insectos/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Temperatura
19.
Artículo en Inglés | MEDLINE | ID: mdl-16511096

RESUMEN

The ervatamins are highly stable cysteine proteases that are present in the latex of the medicinal plant Ervatamia coronaria and belong to the papain family, members of which share similar amino-acid sequences and also a similar fold comprising two domains. Ervatamin A from this family, a highly active protease compared with others from the same source, has been purified to homogeneity by ion-exchange chromatography and crystallized by the vapour-diffusion method. Needle-shaped crystals of ervatamin A diffract to 2.1 A resolution and belong to space group C222(1), with unit-cell parameters a = 31.10, b = 144.17, c = 108.61 A. The solvent content using an ervatamin A molecular weight of 27.6 kDa is 43.9%, with a VM value of 2.19 A3 Da(-1) assuming one protein molecule in the asymmetric unit. A molecular-replacement solution has been found using the structure of ervatamin C as a search model.


Asunto(s)
Cisteína Endopeptidasas/química , Plantas Medicinales/química , Clonación Molecular/métodos , Cristalización/métodos , Cisteína Endopeptidasas/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Volatilización , Difracción de Rayos X
20.
Fitoterapia ; 75(5): 480-93, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15261386

RESUMEN

As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.


Asunto(s)
Apocynaceae/enzimología , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/farmacología , Látex/química , Fitoterapia , Argentina , Cisteína Endopeptidasas/aislamiento & purificación , Humanos , Medicina Tradicional , Tallos de la Planta
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