RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Sapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported. AIM OF THE STUDY: Plant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases. MATERIALS AND METHODS: Protein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically. RESULTS: Seed proteins extract of S. saponaria was evaluated until 100 µg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ââthe biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects. CONCLUSION: The overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs.
Asunto(s)
Daño del ADN/efectos de los fármacos , Extractos Vegetales/farmacología , Extractos Vegetales/toxicidad , Sapindus/química , Semillas/química , Fenómenos Bioquímicos , Muerte Celular/efectos de los fármacos , Cistatinas/química , Cistatinas/aislamiento & purificación , Cistatinas/farmacología , Células Hep G2 , Humanos , Dosificación Letal Mediana , Pruebas de Micronúcleos , Pruebas de Mutagenicidad , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Salmonella typhimurium/efectos de los fármacos , Serina Proteasas/química , Serina Proteasas/aislamiento & purificación , Serina Proteasas/farmacologíaRESUMEN
Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.
Asunto(s)
Colágeno/química , Ficus/química , Látex/química , Proteínas de Plantas/metabolismo , Serina Proteasas/metabolismo , Subtilisinas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Electroforesis en Gel Bidimensional , Ficus/enzimología , Expresión Génica , Calor , Látex/metabolismo , Extractos Vegetales/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Desnaturalización Proteica , Proteolisis , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Proteasas/química , Serina Proteasas/genética , Serina Proteasas/aislamiento & purificación , Especificidad por Sustrato , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/aislamiento & purificaciónRESUMEN
The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers.
Asunto(s)
Bacillus pumilus/enzimología , Bacillus pumilus/genética , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/química , Serina Proteasas/biosíntesis , Serina Proteasas/química , Álcalis/química , Aminoácidos/análisis , Animales , Bacillus pumilus/clasificación , Bacillus pumilus/crecimiento & desarrollo , Fenómenos Bioquímicos , Medios de Cultivo/química , Plumas/química , Plumas/metabolismo , Concentración de Iones de Hidrógeno , Iones/química , Queratinas/química , Queratinas/metabolismo , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/aislamiento & purificación , Aves de Corral , Inhibidores de Proteasas/farmacología , ARN Ribosómico 16S , Serina Proteasas/efectos de los fármacos , Serina Proteasas/aislamiento & purificación , Residuos Sólidos , Solventes/química , Tensoactivos/química , Temperatura , Administración de Residuos/métodosRESUMEN
Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.
Asunto(s)
Caseínas/metabolismo , Gracilaria/enzimología , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Algas Marinas/enzimología , Sulfato de Amonio , Animales , Caseínas/química , Fraccionamiento Químico , Quimosina/metabolismo , Electroforesis en Gel de Poliacrilamida , Gracilaria/química , Concentración de Iones de Hidrógeno , Hidrólisis , Leche/química , Leche/metabolismo , Peso Molecular , Péptido Hidrolasas/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Algas Marinas/química , Serina Proteasas/química , Serina Proteasas/aislamiento & purificación , Serina Proteasas/metabolismo , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
Trichosanthes kirilowii, which is a type of Liana from cucurbitaceous family, possesses many bioactive constituents and therefore has multifarious pharmacological functions. TKP, which is a serine protease extracted from the fruit of Trichosanthes kirilowii, has been reported to possess potential anticancer activity. However, the effects of TKP on cancer cell migration and invasion are still unknown. Here, we reported that TKP could inhibit the migration and invasion abilities of colorectal cancer cells. In addition, the mRNA, protein expression levels, and activities of migration and invasion-related proteins MMP2 and MMP9 were decreased in TKP-treated cells. Mechanistically, TKP treatment repressed Wnt/ß-catenin and Hedgehog/Gli1 signaling cascades. However, the addition of lithium chloride or the transfection of plasmid pcDNA3.1-V5-HisA-Gli1 reversed the impacts of TKP on MMP2, MMP9, cell migration, and invasion. These results indicated that TKP suppressed the migration and invasion of colorectal cancer cells through blocking Wnt/ß-catenin and Hedgehog/Gli1 pathways-mediated MMP2 and MMP9.
Asunto(s)
Adenocarcinoma/patología , Antineoplásicos Fitogénicos/farmacología , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Serina Proteasas/farmacología , Trichosanthes/química , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antineoplásicos Fitogénicos/aislamiento & purificación , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Proteínas Hedgehog/metabolismo , Humanos , Invasividad Neoplásica , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Serina Proteasas/aislamiento & purificación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Trichosanthes/enzimología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , beta Catenina/metabolismoRESUMEN
In recent years, skin reactions such as phytophotodermatitis, contact dermatitis, and other inflammatory responses after contact with chemicals from various plants, e.g., Heracleum mantegazzianum or Hippomane mancinella, are one of the hot topics in phytobiology. Occupational skin inflammation after contact with latices of plants from Euphorbiaceae are common among people who work with plants of this family. Activation of protein kinase C by G protein-coupled receptors such as protease-activated receptors is associated with skin inflammation. In this study, we focused on the inflammatory modulation potential of proteases combined with diterpenes on human skin. Because of its role as a proinflammatory cytokine, we concentrated on the release of IL-8 by fibroblasts and keratinocytes. Therefore, primary human dermal fibroblasts and the HaCaT keratinocytes cell line were used as a model. The results indicated that the combination of the protease mauritanicain from Euphorbia mauritanica and phorbol-12-myristate-13-acetate induced a significantly increased IL-8 release in HaCaT keratinocytes compared to single treatments. The obtained results also suggest that mauritanicain has an anti-inflammatory effect on primary human dermal fibroblasts.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Euphorbia/enzimología , Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Extractos Vegetales/farmacología , Serina Proteasas/farmacología , Acetato de Tetradecanoilforbol/análogos & derivados , Antiinflamatorios no Esteroideos/aislamiento & purificación , Línea Celular , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Interleucina-8/metabolismo , Queratinocitos/metabolismo , Serina Proteasas/aislamiento & purificación , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The characterisation of a serine protease isolated from tamarillo (Solanum betaceum) fruit and its milk casein hydrolysis activity were investigated. Compared with calf rennet, a crude extract from tamarillo exhibited wider caseinolytic activity on sodium caseinate. The purified protease was named "tamarillin" and revealed proteolytic activity toward purified α-, ß- and κ-casein. Similar to calf rennet, tamarillin preferably hydrolysed κ-casein, but, unlike calf rennet, it also displayed high proteolytic activity toward both α- and ß-casein. The major peptide generated from κ-casein by tamarillin was analysed by gel electrophoresis and liquid chromatography mass spectrometry to confirm its molecular mass as 14,290â¯Da. The cleavage site was confirmed by in-gel tryptic digestion and time-of-flight mass spectrometry analysis to be at Asn123-Thr124. This was in contrast to the Phe105-Met106 cleavage site of rennet hydrolysis.
Asunto(s)
Caseínas/metabolismo , Análisis de los Alimentos/métodos , Manipulación de Alimentos/métodos , Frutas/enzimología , Extractos Vegetales/metabolismo , Proteínas de Plantas/metabolismo , Serina Proteasas/metabolismo , Solanum/enzimología , Cromatografía de Fase Inversa , Quimosina/metabolismo , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Peso Molecular , Fragmentos de Péptidos/metabolismo , Extractos Vegetales/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Serina Proteasas/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Especificidad por SustratoRESUMEN
The harnessing of medicinal plants containing a plethora of bioactive molecules may lead to the discovery of novel, potent and safe therapeutic agents to treat thrombosis-associated cardiovascular diseases. A 35 kDa (m/z 34747.5230) serine protease (lunathrombase) showing fibrin(ogen)olytic activity and devoid of N- and O- linked oligosaccharides was purified from an extract of aqueous leaves from L. indica. The LC-MS/MS analysis, de novo sequencing, secondary structure, and amino acid composition determination suggested the enzyme's novel characteristic. Lunathrombase is an αß-fibrinogenase, demonstrating anticoagulant activity with its dual inhibition of thrombin and FXa by a non-enzymatic mechanism. Spectrofluorometric and isothermal calorimetric analyses revealed the binding of lunathrombase to fibrinogen, thrombin, and/or FXa with the generation of endothermic heat. It inhibited collagen/ADP/arachidonic acid-induced mammalian platelet aggregation, and demonstrated antiplatelet activity via COX-1 inhibition and the upregulation of the cAMP level. Lunathrombase showed in vitro thrombolytic activity and was not inhibited by endogenous protease inhibitors α2 macroglobulin and antiplasmin. Lunathrombase was non-cytotoxic to mammalian cells, non-hemolytic, and demonstrated dose-dependent (0.125-0.5 mg/kg) in vivo anticoagulant and plasma defibrinogenation activities in a rodent model. Lunathrombase (10 mg/kg) did not show toxicity or adverse pharmacological effects in treated animals.
Asunto(s)
Anticoagulantes/farmacología , Fibrinolíticos/farmacología , Lamiaceae/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Serina Proteasas/farmacología , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Factores de Coagulación Sanguínea/química , Factores de Coagulación Sanguínea/aislamiento & purificación , Factores de Coagulación Sanguínea/farmacología , AMP Cíclico , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Fibrinólisis/efectos de los fármacos , Fibrinolíticos/química , Fibrinolíticos/aislamiento & purificación , Hemólisis/efectos de los fármacos , Oligosacáridos/química , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Inhibidores de Agregación Plaquetaria/farmacología , Serina Proteasas/química , Serina Proteasas/aislamiento & purificación , Análisis EspectralRESUMEN
A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70â¯kDa. The protease showed optimal activity at pH 11 and 60⯰C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'.
Asunto(s)
Frutas/enzimología , Serina Proteasas/aislamiento & purificación , Solanum/enzimología , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Frutas/genética , Concentración de Iones de Hidrógeno , Peso Molecular , Proteolisis , Análisis de Secuencia de Proteína , Serina Proteasas/genética , Serina Proteasas/metabolismo , Solanum/genética , Subtilisina , TemperaturaRESUMEN
A serine protease was purified from the leaves of Wrightia tinctoria by sequential flow through method comprising screening, optimization, ammonium sulfate precipitation, gel filtration and ion exchange column chromatography. The yield and purification fold obtained were 11.58% and 9.56 respectively. A single band of serine protease was visualized on SDS-PAGE and 2-D gel electrophoretic analyses were revealed with the molecular mass of 38.5 kDa. Serine protease had an optimum pH of 8.0 and was stable at 45°C with high relative protease activity. The addition of metal ions such as Mg2+ and Mn2+ exhibits a high relative activity. Serine protease had a potent antibacterial activity against both Gram-positive and Gram-negative bacteria. A 10 µg/ml of serine protease was tested against S. aureus, M. luteus, P. aeruginosa and K. pneumoniae which had 21, 20, 18 and 17 mm of zone of inhibition respectively. Serine protease from W. tinctoria degrades the peptidoglycan layer of bacteria which was visualized by transmission electron microscopic analysis.
Asunto(s)
Antibacterianos/aislamiento & purificación , Apocynaceae/enzimología , Serina Proteasas/aislamiento & purificación , Sulfato de Amonio/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/ultraestructura , Tampones (Química) , Permeabilidad de la Membrana Celular/efectos de los fármacos , Precipitación Química , Cromatografía de Afinidad , Cromatografía en Gel , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Iones , Metales/farmacología , Pruebas de Sensibilidad Microbiana , Peso Molecular , Extractos Vegetales/química , Hojas de la Planta/enzimología , Inhibidores de Proteasas/farmacología , Solventes/farmacología , Especificidad por Sustrato/efectos de los fármacos , TemperaturaRESUMEN
A protease called Mauritanicain was isolated from the latex of Euphorbia mauritanica L. (Euphorbiaceae) by combining ion exchange chromatography, ultrafiltration, and gel filtration chromatography. It has a high proteolytic activity against casein. The activity was only inhibited by specific serine protease inhibitors, classifying it to the serine protease family. An optimal degradation of the substrate casein takes place at a temperature of 55-65â°C and a pH of 5.5-6.5, and is unstable at pH < 5 and pH > 9. The protease is stable at temperatures from 20-70â°C, whereby the activity decreases drastically to less than 20â% at 75â°C. SDS-PAGE and matrix-assisted laser desorption time-of-flight analysis yielded a molecular weight of 73 kDa; possibly, it is natively present as a non-covalently linked dimer of a higher molecular mass > 132 kDa. Without heat denaturation, a breakdown in fractions of 73 kDa and 52 kDa was observed in SDS-PAGE. Only in some properties it shows a similarity to other characterized proteases in the plant family Euphorbiaceae, such that Mauritanicain can be presented as a new isolated protease.
Asunto(s)
Euphorbia/química , Serina Proteasas/metabolismo , Caseínas/metabolismo , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Látex/química , Peso Molecular , Proteolisis , Serina Proteasas/aislamiento & purificación , Inhibidores de Serina Proteinasa/farmacología , Especificidad por Sustrato , Temperatura , UltrafiltraciónRESUMEN
Plant proteases are capable of performing several functions in biological systems, and their use is attractive for biotechnological process due to their interesting catalytic properties. Bromelia pinguin (aguama) is a wild abundant natural resource in several regions of Central America and the Caribbean Islands but is underutilized. Their fruits are rich in proteases with properties that are still unknown, but they represent an attractive source of enzymes for biotechnological applications. Thus, the proteolytic activity in enzymatic crude extracts (CEs) from wild B. pinguin fruits was partially characterized. Enzymes in CEs showed high proteolytic activity at acid (pH 2.0-4.0) and neutral alkaline (pH 7.0-9.0) conditions, indicating that different types of active proteases are present. Proteolytic activity inhibition by the use of specific protease inhibitors indicated that aspartic, cysteine, and serine proteases are the main types of proteases present in CEs. Activity at pH 3.0 was stable in a broad range of temperatures (25-50 °C) and retained its activity in the presence of surfactants (SDS, Tween-80), reducing agents (DTT, 2-mercapoethanol), and organic solvents (methanol, ethanol, acetone, 2-propanol), which suggests that B. pinguin proteases are potential candidates for their application in brewing, detergent, and pharmaceutical industries.
Asunto(s)
Proteasas de Ácido Aspártico/química , Bromelia/enzimología , Proteasas de Cisteína/química , Frutas/enzimología , Proteínas de Plantas/química , Serina Proteasas/química , Proteasas de Ácido Aspártico/antagonistas & inhibidores , Proteasas de Ácido Aspártico/aislamiento & purificación , Bromelia/química , Proteasas de Cisteína/aislamiento & purificación , Ditiotreitol/química , Pruebas de Enzimas , Frutas/química , Concentración de Iones de Hidrógeno , Cinética , Mercaptoetanol/química , Extractos Vegetales/química , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/aislamiento & purificación , Polisorbatos/química , Inhibidores de Proteasas/química , Proteolisis , Serina Proteasas/aislamiento & purificación , Dodecil Sulfato de Sodio/química , Solventes/químicaRESUMEN
BACKGROUND: The proteases from turmeric species have procoagulant and fibrinogenolytic activity. This provides a scientific basis for traditional use of turmeric to stop bleeding and promote wound healing processes. PURPOSE: Our previous studies revealed that fibrinogenolytic action of crude enzyme fraction of Curcuma aromatica Salisb., was found to be more influential than those of Curcuma longa L., Curcuma caesia Roxb., Curcuma amada Roxb. and Curcuma zedoria (Christm.) Roscoe. Hence, the purpose of this study is to purify and characterize protease from C. aromatica and to explore its role in wound healing process. METHODS: The protease was purified by Sephadex G-50 gel permeation chromatography. Peak with potent proteolytic activity was subjected to rechromatography and then checked for homogeneity by SDS-PAGE and native PAGE. Furthermore purity of the peak was assessed by RP-HPLC and MALDI-TOF. The biochemical properties, type of protease, kinetic studies, fibrinogenolytic, coagulant and fibrinolytic activities were carried out. RESULTS: The two proteolytic peaks were fractionated in gel permeation chromatography. Among these, the peak-II showed potent proteolytic activity with specific activity of 10units/mg/min and named as C. aromatica protease-II (CAP-II). This protein resolved into a single sharp band both in SDS-PAGE (reducing and non-reducing) as well as in native (acidic) PAGE. It is a monomeric protein, showing sharp peak in RP-HPLC and its relative molecular mass was found to be 12.378kDa. The caseinolytic and fibrinolytic activity of CAP-II was completely inhibited by phenylmethylsulfonylfluoride (PMSF). The CAP-II exhibited optimum temperature of 45°C and optimum pH of 7.5. The Km and Vmax of CAP-II was found to be 1.616µg and 1.62units/mg/min respectively. The CAP-II showed hydrolysis of all three subunits of fibrinogen in the order Aα>Bß>γ. The CAP-II exhibited strong procoagulant activity by reducing the human plasma clotting time. It also showed fibrinolytic activity by complete hydrolysis of α-polymer and γ-γ dimer present in fibrin. CONCLUSION: The CAP-II is a novel serine protease from C. aromatica, which has been demonstrated to stop bleeding and initiate wound healing through its procoagulant and fibrin(ogen)olytic activities. Our study demonstrates the possible role of CAP-II, as therapeutic enzyme to stop bleeding at the time of wounding.
Asunto(s)
Curcuma/química , Hemostasis/efectos de los fármacos , Serina Proteasas/aislamiento & purificación , Serina Proteasas/farmacología , Cromatografía Líquida de Alta Presión , Coagulantes/farmacología , Evaluación Preclínica de Medicamentos/métodos , Electroforesis en Gel de Poliacrilamida , Fibrina/metabolismo , Fibrinógeno/metabolismo , Humanos , Cinética , Peso Molecular , Serina Proteasas/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has been traditionally used as a complementary and alternative medicine for cancer treatment by the local communities of Southeast Asia. Despite the continuous research interest in its antiproliferative activity, the identity of the bioactive compound(s) responsible has yet to be determined. This study aims to bridge the gap in existing research literature by using proteomics approach for investigation of the nature of the anticancer substance of L. rhinocerotis. AIM OF THE STUDY: To elucidate the proteome of L. rhinocerotis TM02 sclerotium by protein mass spectrometry and to further isolate and identify the cytotoxic component(s) bearing anticancer potential. MATERIALS AND METHODS: The proteome of L. rhinocerotis sclerotium was analyzed by label-free quantitative shotgun proteomics, using 1D-SDS-PAGE coupled with nano-ESI-LC-MS/MS based on the availability of its genome-sequence database. The cytotoxicity of L. rhinocerotis sclerotial extracts against human breast adenocarcinoma cells (MCF7) were assessed by MTT cytotoxicity assay prior to successive purification steps by a combination of gel filtration chromatography, ammonium sulfate precipitation, and anion exchange chromatography. Bioactive compound(s) in the extracts was identified by shotgun proteomics and N-terminal protein sequencing. RESULTS: Several proteins with interesting biological activities including lectins, fungal immunomodulatory proteins, and several antioxidant proteins were identified from the proteome of L. rhinocerotis. A cytotoxic protein fraction (termed F5) which was partially purified from its sclerotial cold water extract F5 shows two distinct bands of 31 and 36 kDa in reducing SDS-PAGE and exhibited potent selective cytotoxicity against MCF7 cells with IC50 value of 3.00 ± 1.01 µg/ml. Both bands were identified to be serine protease by LC-MS/MS analysis. Phenylmethylsulfonyl fluoride, a specific serine protease inhibitor, inhibited both the proteolytic activity and cytotoxicity of F5, suggesting that the cytotoxicity of F5 is related to its protease activity. CONCLUSIONS: This study provides the first comprehensive and semi-quantitative profiling of the proteome of L. rhinocerotis sclerotium. Further investigation into its selective cytotoxicity shows that a serine protease-like protein, termed F5, may be targeted for new anticancer agent development.
Asunto(s)
Agaricales/genética , Antineoplásicos/farmacología , Proteómica/métodos , Serina Proteasas/genética , Agaricales/enzimología , Antineoplásicos/aislamiento & purificación , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Línea Celular Tumoral , Bases de Datos Genéticas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Factores Inmunológicos/aislamiento & purificación , Factores Inmunológicos/farmacología , Lectinas/aislamiento & purificación , Lectinas/farmacología , Medicina Tradicional de Asia Oriental , Serina Proteasas/aislamiento & purificación , Serina Proteasas/farmacología , Sales de Tetrazolio , TiazolesRESUMEN
Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.
Asunto(s)
Venenos de Crotálidos/enzimología , Crotalus , Serina Proteasas/aislamiento & purificación , Serina Proteasas/metabolismo , Animales , Coagulación Sanguínea , Bovinos , Clonación Molecular , Cobre/metabolismo , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Fibrinógeno/metabolismo , Fibrinopéptido A/metabolismo , Expresión Génica , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serina Proteasas/química , Serina Proteasas/genética , Temperatura , Zinc/metabolismoRESUMEN
For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Dípteros/enzimología , Proteínas de Insectos/farmacología , Serina Proteasas/farmacología , Animales , Factores de Coagulación Sanguínea/efectos de los fármacos , Factores de Coagulación Sanguínea/metabolismo , Pruebas de Coagulación Sanguínea , Proteína Inhibidora del Complemento C1/metabolismo , Proteína Inhibidora del Complemento C1/farmacología , Desbridamiento , Dípteros/crecimiento & desarrollo , Activación Enzimática/efectos de los fármacos , Factor XIIa/biosíntesis , Heces , Proteínas de Insectos/aislamiento & purificación , Calicreínas/sangre , Larva/enzimología , Nefelometría y Turbidimetría , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Serina Proteasas/aislamiento & purificación , Tromboelastografía , Cicatrización de HeridasRESUMEN
A new, direct-acting chymotrypsin-like fibrinolytic serine protease was purified from Petasites japonicus, a medicinal herb. The molecular mass of the discovered enzyme was estimated to be 40.0 kDa as determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, fibrin zymography, and gel filtration chromatography. The N-terminal sequence of the purified enzyme was determined to be GQEDHFLQVSLTSA. The proteolytic activity of the enzyme was found to be inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(amidinophenyl) methanesulfonyl fluoride. An assay of enzyme activity on fibrin plates revealed that it could hydrolyze the fibrin directly. The enzyme displayed a potent fibrin(ogen)olytic activity, hydrolyzing the Aα-, α-, and Bß-subunits of the human fibrinogen. The enzyme prolonged activated partial thromboplastin time and had little effect on prothrombin time. It prevented carrageenan-induced thrombus formation in mouse tails and did not increase the bleeding time. Our findings indicate that the extracted enzyme we present here has the potential for clinical use as an agent for the treatment of thrombosis.
Asunto(s)
Fibrinólisis , Petasites/enzimología , Serina Proteasas/aislamiento & purificación , Amidas/metabolismo , Secuencia de Aminoácidos , Animales , Anticoagulantes/farmacología , Antitrombinas/farmacología , Electroforesis en Gel de Poliacrilamida , Fibrinógeno/metabolismo , Fibrinólisis/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Análisis de Secuencia de Proteína , Serina Proteasas/química , Trombosis/patologíaRESUMEN
Leishmaniasis, a neglected tropical disease (NTD) causes major health problems in the tropical and subtropical world. Most of the antileishmanial modern therapies with different formulations of pentavalent antimonials, Miltefosine, Amphotericin B etc. are not satisfactory in recent times due to high toxicity to the host and present rising strain resistance issues. So there is an urgent need to develop new, safe and cost-effective drugs against leishmaniasis. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The prominent roles played by Leishmania proteases in the virulence of this parasite make them very promising targets for the development of current therapeutics of leishmaniasis. As part of a search for novel drugs, we have evaluated in vitro anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx) obtained from potato tuber. The extract (PTEx) was prepared by sodium bisulfite fractionation and inhibitors were identified by reverse zymography. Inhibition study of PTEx in gelatin-zymogram and spectrophotometric assay using BApNA and BTpNA as substrate reveal its strong inhibitory activity against trypsin as well as serine proteases present in cell lysate of Leishmania donovani infective strain. The in vitro MTT based colorimetric assay as well as ex vivo L. donovani infected macrophages showed reduced parasite viability and intracellular parasite load with IC50 = 312.5 ± 0.1 µg/ml and IC50 82.3 ± 0.2 µg/ml of PTEx respectively in a concentration dependent manner. This anti-leishmanial effect was also preceded by PTEx induced acute formation of ROS and prolonged NO generation. The PTEx has no significant cytotoxic effect on host macrophages. So taken together, these findings indicate that PTEx has promising leishmanicidal effect and thus this study provides a new perspective of natural serine protease inhibitor from potato tuber on the development of new drug against leishmaniasis.
Asunto(s)
Leishmania donovani/enzimología , Leishmaniasis Visceral/tratamiento farmacológico , Extractos Vegetales/farmacología , Inhibidores de Proteasas/farmacología , Solanum tuberosum/química , Animales , Cricetinae , Electroforesis , Leishmania donovani/efectos de los fármacos , Mesocricetus , Ratones , Ratones Endogámicos BALB C , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Proteasas/uso terapéutico , Serina Proteasas/aislamiento & purificaciónRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Wrightia tinctoria R. Br. (Apocyanaceae) is a folk medicinal plant known to have immunomodulatory, anti-inflammatory and antihemorrhagic potential. Wrightia tinctoria latex is used for treatment of various clinical conditions including psoriasis, blisters, mouth ulcers, and extensively for topical application on fresh wounds to promote accelerated healing. AIMS OF THE STUDY: To investigate the wound healing potential of Wrightia tinctoria latex proteases using a mouse model. MATERIALS AND METHODS: Proteolytic activity of Wrightia tinctoria latex proteases (WTLP) was determined on various substrates (casein, gelatin and collagen (type-I and IV)). The thermal stability and the class of proteases present in WTLP were determined using heat treatment and specific protease inhibitors, respectively. Excision wound model in mice was used to evaluate the healing potential of WTLP application (twice daily, 10mg/kg). Neosporin, a standard drug, was used for comparison. The progression of healing was monitored using physical (wound contraction), biochemical (collagen content, catalase and MMP activity) and histological examinations. RESULTS: WTLP contains thermostable serine proteases, which are completely inhibited by PMSF. WTLP showed strong caseinolytic, gelatinolytic and collagenolytic activity. The excision wound healing rate upon WTLP treatment was significantly higher than (>2-fold) the control group (49% vs. 18%, (**)p<0.01) on day 3 and throughout the study. PMSF pre-treated and heat denatured WTLP failed to promote wound healing. In addition, serial biochemical analysis of the granulation tissue demonstrated 1.5-fold more (2444 ± 100 vs. 1579 ± 121 µg/100mg tissue) hydroxyproline content and 5.6-fold higher catalase activity (16.7 ± 1.3 vs. 3 ± 0.3 units/mg) compared to controls. Further, the enhanced collagen content and matrix metalloproteinase activity correlated with wound contraction rate following WTLP and Neosporin treatment. Histological analysis on day 9 confirmed complete epithelialization, re-establishment of skin structure and accelerated wound healing following WTLP treatment. CONCLUSIONS: The thermostable serine proteases of Wrightia tinctoria latex are directly involved in the wound healing process. Our findings provide a biochemical basis for the role of WTLP in the enhancement of wound healing. The study supports traditional topical application of Wrightia tinctoria latex on fresh wounds to promote accelerated healing.
Asunto(s)
Apocynaceae/química , Látex , Serina Proteasas/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Heridas Penetrantes/tratamiento farmacológico , Administración Tópica , Animales , Apocynaceae/enzimología , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Estabilidad de Enzimas , Etnofarmacología , Femenino , Calor , India , Masculino , Ratones , Inhibidores de Proteasas/farmacología , Serina Proteasas/aislamiento & purificación , Serina Proteasas/farmacología , Heridas Penetrantes/patologíaRESUMEN
A 34 kDa serine protease, designated as hirtin, with fibrinolytic activity was purified to homogeneity from the latex of Euphorbia hirta by the combination of ion exchange and gel filtration chromatography. The N-terminal sequence of hirtin was found to be YAVYIGLILETAA/NNE. Hirtin exhibited esterase and amidase activities along with azocaseinolytic, gelatinolytic, fibrinogenolytic and fibrinolytic activities. It preferentially hydrolyzed Aα and α-chains, followed by Bß and ß, and γ and γ-γ chains of fibrinogen and fibrin clot respectively. The optimum pH and temperature for enzyme activity was found to be pH 7.2 and 50 °C respectively. Enzymatic activity of hirtin was significantly inhibited by PMSF and AEBSF. It showed higher specificity for synthetic substrate p-tos-GPRNA for thrombin. The CD spectra of hirtin showed a high content of ß-sheets as compared to α-helix. The results indicate that hirtin is a thrombin-like serine protease and may have potential industrial and therapeutic applications.