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Effects of Mucuna pruriens Protease Inhibitors on Echis carinatus Venom
Abstract
The medicinal plant Mucuna pruriens (MP), with anti-snake properties has been reported to contain kunitz-type trypsin inhibitor. This study was done to further evaluate the protease inhibitory potential of gpMuc, a glycoprotein and other protein fractions from MP seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results obtained showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease recognizing epitope on one of its isoforms. The whole protein extract of MP seeds inhibited prothrombin activation by ecarin and whole Echis carinatus venom, and also thrombin like activity using chromogenic assay.
... Furthermore, it has been reported to stimulate the synthesis of antibodies capable of cross-reacting with venomous proteins and inhibits the venom proteolytic apparatuses, thus mitigating toxicity (Guerranti et al., 2004;Kumar et al., 2016). This immunogenic glycoprotein (gpMuc) has a molecular weight of 20.3 to 28.7 kDa and pI of 4.8 to 6.5 (Di Patrizi et al., 2006;Hope-Onyekwere, 2012) and has Seven (7) different N-terminal isoforms, some of which possess structural similarities to PLA2 of venoms (Lucia et al., 2012). Recent investigations by Kumar et al. (2016) substantiated that a specific purified protein from M. pruriens code-named MP-4 (20.9 kDa) reacts with antibodies against Echiscarinatus venom, though contrarily, the MP-4 protein exhibited no direct protease inhibitory action. ...
Snakes are primarily venomous animals that bite when frightened, which can be lethal. This is because snake venom is one of the most active biological fluids containing a wide range of peptides and proteins that can induce several effects, including hemo-, neuro-, cyto-and myotoxic effects, consequently becoming deleterious to life if untreated. Although snakes are found on almost all continents, the rural communities in sub-Saharan Africa are the most affected by snakebites, mainly due to increased human-snake interactions forced by their socioeconomic status and agricultural or rural practices. Consequently, this recently prompted the World Health Organisation to enlist snakebites envenoming among the category-A neglected tropical diseases with an estimated annual death of 7,300 in sub-Saharan Africa. Aside from mortality, snakebite envenomation also causes permanent disabilities in humans and a heavy burden on livestock, creating economic hardship for the already impoverished communities. Several animal-derived antivenoms have been developed for treating snakebites and wounds; they effectively attenuate venom-related toxicity, tissue necrosis, and deaths. However, despite the efficacy of these antivenoms, several issues, such as problems in production and distribution, exorbitant prices, and adverse effects of the antivenoms, have challenged their practical use in sub-Saharan Africa. This review highlights the challenges that make conventional antivenoms unavailable to prone populations. We also discuss the plants used in the treatment of snake bites laying emphasis on Mucuna pruriens (Velvet bean) and Allium sativum (Garlic) as the two most studied medicinal plants. The progress and bottlenecks of using herbal antivenoms as alternatives in treating snakebite envenomation in sub-Saharan Africa are also discussed.
... These effects may be more significant in individuals who are sensitive to Mucuna pruriens. It has also been indicated that casual skin contact with the plant pod produces erythema and pruritic macular lesion caused by the mucunain protease (Hoe-Onyekwere, 2012)In addition, the rubbing of the seed cover against the skin may facilitate the dermal absorption of the biologically active compounds. ...
... One of the proteins present in the seed extract is a multiform glycoprotein (gpMuc) of apparent molecular weight 20-28 kDa. N-terminal sequences of seven glycosylated isoforms of this protein show the conserved signature sequence of Kunitz-type protease inhibitors (27)(28). This protein can inhibit proteolytic components of snake venom and thus may provide direct protection against toxic effects of snakebite. ...
Mortality due to snakebite is a serious public health problem and available therapeutics are known to induce debilitating
side effects. Traditional medicine suggests that seeds of Mucuna pruriens can provide protection against the effects of snakebite.
Our aim is to identify the protein/s that may be important for snake venom neutralization and elucidate their mechanism of
action. To this end, we have identified and purified a protein from Mucuna pruriens, which we have named MP-4. The full-length
polypeptide sequence of MP-4 was obtained through N-terminal sequencing of peptide fragments. Sequence analysis suggested
that the protein may belong to the Kunitz-type protease inhibitor (KTPI) family and therefore may potentially neutralize the
proteases present in snake venom. Using various structural and biochemical tools coupled with in vivo assays, we are able
to show that MP-4 does not afford direct protection against snake venom because it is actually a poor inhibitor of serine
proteases. Further experiments showed that antibodies generated against MP-4 cross-react with the whole venom and provide
protection to mice against Echis carinatus snake venom. This study shows that the MP-4 contributes significantly to the snake
venom neutralization activity of Mucuna pruriens seeds through an indirect antibody-mediated mechanism.
... A proteomic analysis indicated that the MPE pre-treatment elicited the activation of counterbalancing processes to compensate for the imbalances caused by E.carinatus venom 6 . Subsequently, it was demonstrated that the direct protective effect of MPE against the venom was due to a Kunitz-type trypsin inhibitor 7,8 . On the other hand, it was demonstrated that the protective effect of MPE-pretreatment against cobra venom lethality involved a direct cardio-protective action, in addition to the known immunological mechanism 9 . ...
Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.
... According to their sequences, we can group the isoforms at positions 1, 2, and 4 on the gel, which are identical in 12/12 aa. The isoform at position 3 is identical to those aforementioned, with regard to the first 10 aa, and those at positions 5, 6, and 7 differ from those at positions 1,2 and 4 by just 3 aa (Guerranti et al., 2002;Scirè et al., 2011;Hope-Onyekwere et al., 2012). On the other hand, the direct inhibitory action of MPE is probably caused by L-dopa, the main bioactive component, which acts in synergy with other compounds. ...
Mucuna pruriens (Fabaceae) is an established herbal drug used for the management of male infertility, nervous disorders, and also as an aphrodisiac. It has been shown that its seeds are potentially of substantial medicinal importance. The ancient Indian medical system, Ayurveda, traditionally used M. pruriens, even to treat such things as Parkinson's disease. M. pruriens has been shown to have anti-parkinson and neuroprotective effects, which may be related to its anti-oxidant activity. In addition, anti-oxidant activity of M. pruriens has been also demonstrated in vitro by its ability to scavenge DPPH radicals and reactive oxygen species. In this review the medicinal properties of M. pruriens are summarized, taking in consideration the studies that have used the seeds extracts and the leaves extracts.
... M. pruriens seeds have been investigated for their many chemical and pharmacological properties: antidiabetic, antiepileptic, antimicrobial, aphrodisiac [6] and anti-Parkinson activity [7], as well as oral prophylaxis for snake bite [8][9][10]. Many properties of M. pruriens are due to the presence of protease inhibitors [11,12], which may be involved in plant defences against pests and diseases [13], or to seed storage proteins [14], probably contained in protein bodies. For all these reasons, the seeds of this species are worthy of research. ...
Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5-12 μm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.
El cultivo de guanábana (Annona muricata L.) es uno de los más populares en Colombia, puesto que su fruto es muy consumido y requiere de buenos cuidados para su comercialización, en los últimos años se ha ido incrementando el área sembrada. La aplicación de productos químicos a estos árboles para la disminución de la enfermedad que los afecta, antracnosis (Colletotrichum gloeosporoides) es una constante; por lo tanto este proyecto investigó sobre otras alternativas probando productos orgánicos elaborados a base de extractos vegetales o biofertilizantes que puedan disminuir y controlar la incidencia y severidad de esta enfermedad, con el fin de reemplazar los agroquímicos que dejan residualidad y pueden ser perjudiciales para la salud humana. Para estas pruebas se establecieron los siguientes tratamientos: Mucuna pruriens (mucuna) (T1), Datura stramonium (borrachero) (T2) y un biofertilizante (T3) para comparar la severidad de la enfermedad contra un testigo (T0); se aplicó un diseño completamente al azar, cuatro tratamientos, tres replicaciones con tres unidades experimentales, se realizaron evaluaciones a los siete, doce y dieciocho días después de la aplicación de estos productos. El porcentaje de disminución con respecto a la severidad inicial que se tomó como cero (0), arroja que los productos que dieron un resultado positivo (P<0.05) en las aplicaciones de manera progresiva contra la enfermedad antracnosis fueron: el extracto con la planta Mucuna pruriens y el biofertilizante siendo de 78.35 Vs 18.36 y 50.81 Vs 20%, respectivamente comparando la severidad inicial con la presentada a los 18 días después de la aplicación de productos. Se concluye que los mejores tratamientos fueron el de Mucuna pruriens seguido del biofertilizante.
A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 10–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.
Evolution of proteinase inhibitor diversity in leguminous plants of tropical rainforests is under immense pressure from the regular upregulation of proteolytic machinery of their pests. The present study illustrates the isolation and bioinsecticidal potency of a serine proteinase inhibitor from the seeds of Caesalpinia bonduc (CbTI), inhabiting Great Nicobar Island, India. Following initial fractionation by ammonium sulfate precipitation, CbTI was purified to homogeneity by ion exchange, gel filtration and trypsin affinity chromatography. SDS-PAGE of gel filtrated CbTI showed a couple of proteins CbTI-1 ( approximately 16kDa) and CbTI-2 (20kDa) under non-reducing conditions, which subsequent to trypsin affinity chromatography yielded only CbTI-2. Both Native PAGE as well as iso-electric focusing showed 2 iso-inhibitors of CbTI-2 (pI values of 5.35 and 4.6). CbTI exhibited tolerance to extremes of temperatures (0-60 degrees C) and pH (1-12). A 1:1 stoichiometric ratio was noted during CbTI-2-trypsin complex formation, which was absent on binding with chymotrypsin. Further, SDS-PAGE analysis also showed that CbTI-1 has affinity only towards chymotrypsin, whereas both trypsin and chymotrypsin formed complexes with CbTI-2. Dixon plot analysis of CbTI-2 yielded inhibition constants (K(i)) of 2.75 x 10(-10)M and 0.95 x 10(-10)M against trypsin and chymotrypsin activity respectively. Preliminary investigations on the toxicological nature of CbTI revealed it to be a promising bioinsecticidal candidate.
A Bowman-Birk-type trypsin inhibitor (TcTI) was purified from seeds of Torresea cearensis, a Brazilian native tree of the Papilionoideae sub-family of Leguminosae. Three forms of the inhibitor were separated by anion exchange chromatography. The major form with 63 amino acids was entirely sequenced; it shows a high structural similarity to the Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of the inhibitor are a lysine residue at position 15 and a histidine at position 42 as identified by alignment to related inhibitors, direct chemical modification and specific enzymatic degradation. Immunoprecipitation with antibodies raised in rats is reduced significantly if TcTI is complexed with chymotrypsin and, to a lesser degree, if complexed with trypsin. TcTI forms a ternary complex with trypsin and chymotrypsin. The binary complexes with trypsin or chymotrypsin were isolated by gel filtration. Dissociation constants of the complexes with trypsin, plasmin, chymotrypsin, and factor XIIa are 1, 36, 50, 1450 nM, respectively; human plasma kallikrein, human factor Xa, porcine pancreatic kallikrein and bovine thrombin are not inhibited. TcTI prolongs blood clotting time of the contact phase activation pathway by inhibition of FXIIa.
A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30-60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 x 10(-9) M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.
The seed powder of the leguminous plant, Mucuna pruriens has long been used in traditional Ayurvedic Indian medicine for diseases including parkinsonism. We have assessed the clinical effects and levodopa (L-dopa) pharmacokinetics following two different doses of mucuna preparation and compared them with standard L-dopa/carbidopa (LD/CD).
Eight Parkinson's disease patients with a short duration L-dopa response and on period dyskinesias completed a randomised, controlled, double blind crossover trial. Patients were challenged with single doses of 200/50 mg LD/CD, and 15 and 30 g of mucuna preparation in randomised order at weekly intervals. L-dopa pharmacokinetics were determined, and Unified Parkinson's Disease Rating Scale and tapping speed were obtained at baseline and repeatedly during the 4 h following drug ingestion. Dyskinesias were assessed using modified AIMS and Goetz scales.
Compared with standard LD/CD, the 30 g mucuna preparation led to a considerably faster onset of effect (34.6 v 68.5 min; p = 0.021), reflected in shorter latencies to peak L-dopa plasma concentrations. Mean on time was 21.9% (37 min) longer with 30 g mucuna than with LD/CD (p = 0.021); peak L-dopa plasma concentrations were 110% higher and the area under the plasma concentration v time curve (area under curve) was 165.3% larger (p = 0.012). No significant differences in dyskinesias or tolerability occurred.
The rapid onset of action and longer on time without concomitant increase in dyskinesias on mucuna seed powder formulation suggest that this natural source of L-dopa might possess advantages over conventional L-dopa preparations in the long term management of PD. Assessment of long term efficacy and tolerability in a randomised, controlled study is warranted.
Leguminous plants in the tropical rainforests are a rich source of proteinase inhibitors and this work illustrates isolation of a serine proteinase inhibitor from the seeds of Archidendron ellipticum (AeTI), inhabiting Great Nicobar Island, India. AeTI was purified to homogeneity by acetone and ammonium sulfate fractionation, and ion exchange, size exclusion and reverse phase chromatography (HPLC). SDS-PAGE of AeTI revealed that it is constituted by two polypeptide chains (alpha-chain, M(r) 15,000 and beta-chain, M(r) 5000), the molecular weight being approximately 20 kDa. N-terminal sequence showed high homology with other serine proteinase inhibitors belonging to the Mimosoideae subfamily. Both Native-PAGE as well as isoelectric focussing showed four isoinhibitors (pI values of 4.1, 4.55, 5.27 and 5.65). Inhibitory activity of AeTI remained unchanged over a wide range of temperatures (0-60 degrees C) and pH (1-10). The protein inhibited trypsin in the stoichiometric ratio of 1:1, but lacked similar stoichiometry against chymotrypsin. Also, AeTI-trypsin complex was stable to SDS unlike the SDS unstable AeTI-chymotrypsin complex. AeTI, which possessed inhibition constants (K(i)) of 2.46 x 10(-10) and 0.5 x 10(-10)M against trypsin and chymotrypsin activity, respectively, retained over 70% of inhibitory activity after being stored at -20 degrees C for more than a year. Initial studies on the insecticidal properties of AeTI indicate it to be a very potent insect anti-feedant.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
This paper reports the preliminary characterization of seed protein fractions from seven varieties of velvet beans (Mucuna pruriens) grown in Nigeria, using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Three of the most abundant polypeptides, with approximate Mr of 23, 26 and 30 kDa, respectively, were further separated by preparative native-PAGE. N-terminal sequencing revealed the presence of the consensus sequence DDREPV-DT–PL that is also present in the soybean Kunitz-type trypsin inhibitor. The albumin fraction was also shown to contain both trypsin and chymotrypsin inhibitors through enzyme inhibitor assays. Western analysis using antibodies, raised against a representative, 23 kDa polypeptide, indicated that this protein species accumulates exclusively during seed development, suggesting a role in seed storage. Haemagglutination assays using rabbit erythrocytes failed to detect the presence of lectins. The results are discussed within the context of the role of lectins and protease inhibitors in storage and plant defence. The findings are also relevant in view of the toxic and antimetabolic effects of these proteins, which determine the acceptability and adoption of velvet beans as animal and human feed.
Alcohol Extracts Of Leaf and Fruit Trichome Of Mucuna Pruriens Dc, An African Traditional Plant, Were Screened For Analgesic and Antipyretic Activity In The Rat. The Extracts Were Administered By Gavage and Found To Increase The Pain Threshold and Decrease Body Temperature, When Pyrexia Was Induced By Injecting A Yeast Suspension. The Extracts Also Showed Anti-Inflammatory Activity, As They Were Able To Inhibit Carrageenin-Induced Edema. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Eighteen proteases were isolated from six hemorrhagic venoms of snakes belonging to the families of Crotalidae and Viperidae. According to their actions, they are classified as thrombin-like enzymes, alpha-fibrinogenases, beta-fibrinogenases, Factor X activator, prothrombin activator, hemorrhagins and esterases. Thrombin-like enzymes, beta-fibrinogenases, hemorrhagins and esterase hydrolyzed Phe-Pip-Arg-pNA (S-2238, substrate for thrombin) more strongly than CBZ-Ile-Glu-Gly-Arg-pNA (S-2222, substrate for Factor Xa), CBZ-Phe-Val-Arg-pNA (B-7632) or CBZ-Pro-Phe-Arg-pNA (B-2133). Thrombin-like enzymes, beta-fibrinogenase and esterase hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine ethyl ester. S-2238 is the most susceptible chromogenic substrate for most venom proteases. Thrombin-like enzymes degraded prothrombin molecule progressively down to prethrombin 2 while alpha- and beta-fibrinogenases degraded it only to prethrombin 1. Factor X activator of Vipera russelli venom and esterase of T. mucrosquamatus venom did not have any effect on prothrombin. Thus, the effects of venom proteases on prothrombin are not parallel to their amidolytic or esterolytic effects.
Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40-50%) alpha-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.
Bowman-Birk inhibitor was partially hydrolyzed with chymotrypsin [EC 3. 4. 4. 5] at pH 3.0, and the subsequent reduction, S-carboxymethylation and gel filtration of the product yielded two fragments. One fragment containing a methionine residue was further degraded into two fragments with cyanogen bromide. By the amino acid determination and the terminal sequence analysis of these three fragments as well as of the whole protein, the tryptic peptides were aligned into the complete amino acid sequence of the inhibitor.
The anti-chymotrypsin and the anti-trypsin sites of the inhibitor were also identified by the limited hydrolysis with these proteinases.
The sequence of this inhibitor was found to be very similar to that of lima bean inhibitor IV (Tan and Stevens, 1971), and consisted of two homologous peptide regions, one includes the inhibitory site for trypsin [EC 3. 4. 4. 4] and the other that for chymotrypsin.
Comparison with other leguminosae inhibitors was also discussed.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A review has been presented of the biochemistry and pharmacology of a class of natural products, the flavonoids. These substances which are widely distributed in the plant kingdom and present in considerable quantities in common food products, spices and beverages have in a concentrated form (Propolis) been used since ancient times by physicians and laymen to treat a great variety of human diseases but they have yet to pass the tests of modern, controlled, clinical experimentation. An attempt has been made to present the fundamental evidence from the basic biological sciences which is required to stimulate the interest of the clinicians in this new field. The few existing reports on the careful pharmacodynamic, pharmacokinetic and clinical studies which have been made have been summarized to provide a basis for a full-scale investigation of the therapeutic potential of flavonoids.
A technique is described to detect the activity of protease inhibitors present in sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAG) containing a copolymerized enzyme substrate. The method involved (1) incorporation of substrate (gelatin or casein) into the SDS-PAG at the time of casting; (2) electrophoresis of the protease inhibitors in the presence of SDS; (3) removal of SDS by washing the gel in 2.5% (w/v) Triton X-100; (4) incubation of the gels in a solution containing the proteolytic enzyme at 37 degrees C for 16 h; and (5) staining undigested substrate with amido black. Standard inhibitors such as bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (SBTI), alpha 1-antitrypsin inhibitor, and a protease inhibitor derived from human articular cartilage have been examined by this method and displayed sharp inhibition bands when the gels were treated with bovine trypsin, chymotrypsin, or other enzymes. The technique cannot be used for precise quantification of protease inhibitors. However, there is a relationship between the concentration of inhibitor used and the intensity of staining. By this means, it was possible to estimate the smallest amount of inhibitor that could be detected (against a particular enzyme) under a given set of conditions. Inhibition was detected when 10 ng of SBTI or 20 ng of BPTI were applied to the gels; human alpha 1-protease inhibitor could be detected at a level of 2-3 micrograms. The technique was used to investigate the effectiveness of the human cartilage inhibitor against a variety of proteolytic enzymes, including thermolysin, Pronase, neutral protease, elastase, protease VII, pepsin, bacterial collagenase, protease IV, and papain.
Two proteinase inhibitors, DE-1 and DE-3, were purified from Erythrina latissima seeds. Whereas DE-1 inhibits bovine chymotrypsin and not bovine trypsin, DE-3 inhibits trypsin but not chymotrypsin. The molecular weights and the amino acid compositions of the two inhibitors resemble the corresponding properties of the Kunitz-type proteinase inhibitors. The N-terminal primary structure of DE-3 showed homology with soybean trypsin inhibitor (Kunitz) and also with the proteinase inhibitors (A-II and B-II) from Albizzia julibrissin seed.
A proteinase inhibitor (DE-1) from the seed of Peltophorum africanum (Weeping wattle) was purified by chromatographic procedures involving Sephadex G-50, DEAE-cellulose and DEAE-Sepharose. It comprises 162 amino acid residues (molecular mass 18 000 Da) including 4 half-cystine residues and resembles the Kunitz-type proteinase inhibitors. The N-terminal primary structure of DE-1 showed homology with Kunitz soybean trypsin inhibitor and also with the proteinase inhibitors from Albizzia julibrissin and Erythrina latissima seeds. The inhibitor stoichiometrically inhibited trypsin and also alpha-chymotrypsin in the molar ratio of 1:1 and represents, therefore, a Kunitz-type double-headed proteinase inhibitor.
After having established the basic protocol of two-dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531-546), some critical parameters of the actual IPG-Dalt protocols as well as the results obtained with horizontal and vertical second-dimensional sodium dodecyl sulfate-electrophoresis are demonstrated and discussed.
Schizolobium parahyba seed chymotrypsin inhibitor (SPC) is a protein with M(r) of 20,000 and four half-cystine residues and no free thiol group. SPC is stable at temperatures up to 75 degrees at pH 7 but gradually loses activity when kept at 95 degrees for 1 hr and total inactivation occurs after 5 hr. Amino acid analysis shows a high content of glycine, aspartate, glutamate and alanine residues. A pI of 4.52 predicted from the amino acid content agrees with experimental results. A stable binary complex with M(r) of 45,000, Ki = 5.85 x 10(-8) M and molar ratio of 1:1 is formed between SPC and chymotrypsin. The determined single N-terminal sequence of SPC shows homology with Kunitz type soybean trypsin inhibitors.
A list of flowering plants used for the treatment of snakebite has been complied from a variety of literature sources. Details of the geographical area and parts used are given and the basis for the reputed activity discussed. Methods of testing the reputed activity of such plants are reviewed and the identity and mode of action of the chemical substances which might be responsible is discussed for some of the plants listed.
A trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an alpha-chain consisting of 134 residues and a beta-chain made up of 40 residues. The inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. The reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.
A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.
A protein proteinase inhibitor (PI) has been purified from pigeonpea Cajanus cajan (L.) PUSA 33 variety by acetic-acid precipitation, salt fractionation and chromatography on a DEAE-Cellulose column. The content of inhibitor was found to be 15 mg/20 g dry weight of pulse. The molecular weight of the inhibitor as determined by SDS-PAGE under reducing conditions was found to be about 14,000. It showed inhibitory activity toward proteolytic enzymes belonging to the serine protease group, namely trypsin and alpha-chymotrypsin. The inhibitory activity was stable over a wide range of pH and temperatures. Estimation of sulfhydryl groups yielded one free cysteine and at least two disulfide linkages. N-terminal sequence homology suggests that it belongs to the Kunitz inhibitor family. Structural analysis by circular dichroism shows that the inhibitor possesses a largely disordered structure.
In a previous paper we demonstrated that extracts of Mucuna pruriens seeds (MPE) protect mice against Echis carinatus venom (EV) by an immunological mechanism. In this paper we demonstrate that the MPE immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc) whose immunogenic properties mainly reside in its glycan-chains. The glycoprotein was purified from the protein extract of M. pruriens seeds using Concanavalin A affinity chromatography. Using 2-D gel electrophoresis it separated into seven isoforms having MWs in the range from 20.3 to 28.7 kDa and pIs from 4.8 to 6.5. N-terminal sequencing of these spots revealed close similarity since all of them contained the consensus sequence DDREPV-DT found in soybean Kunitz-type trypsin inhibitor. We suggest that gpMuc contains both N- and O-glycans. Mild alkaline treatment but not PNGase F led to loss of reactivity, indicating that O-glycans are probably involved in the antigenicity of gpMuc.
Envenomations due to snake bites are commonly treated by parenteral administration of horse or sheep-derived polyclonal antivenoms aimed at the neutralization of toxins. However, despite the widespread success of this therapy, it is still important to search for different venom inhibitors, either synthetic or natural, that could complement or substitute for the action of antivenoms. Several plants have been utilized in folk medicine as antiophidian. However, only a few species have been scientifically investigated and still less had their active components isolated and characterized both structurally and functionally. This article presents a review of plants showing neutralizing properties against snake venoms which were assayed in research laboratories, correlating them with ethnopharmacological studies, as (i) the part of the plant used as antidote, (ii) its respective genus and family and (iii) inhibition of the main pharmacological, toxic and enzymatic activities of snake venoms and isolated toxins. Protective activity of many of these plants against the lethal action of snake venoms has been confirmed by biological assays. Compounds in all of them belong to chemical classes capable of interacting with macromolecular targets (enzymes or receptors). Popular culture can often help to guide scientific studies. In addition, biotechnological application of these inhibitors, as helpful alternative or supplemental treatments to serum therapy, and also as important models for synthesis of new drugs of medical interest, needs to be better oriented and scientifically explored.
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.
Inga laurina is a tree that belongs to the Mimosoideae sub-family of the Leguminosae. A protein inhibitor of trypsin (ILTI) was isolated from its seeds by ammonium sulphate precipitation, ion-exchange chromatography and rechromatography on an HiTrap Q ion-exchange column. By SDS-PAGE, ILTI yielded a single band with a Mr of 20 kDa with or without reduction. ILTI was found to be a single polypeptide chain containing 180 amino acids, the sequence of which was clearly homologous to the Kunitz family of serine protease plant protein inhibitors, and it also showed significant similarity to the seed storage proteins, sporamin and miraculin. However, ILTI displayed major differences to most other Kunitz inhibitors in that it contained only one disulfide bridge, and did not have two polypeptide chains as for the majority of other Kunitz inhibitors purified from Mimosoideae species. ILTI inhibited bovine trypsin with an equilibrium dissociation constant (K(i)) of 6 x 10(-9)M, but did not inhibit chymotrypsin, papain and alpha-amylase. Its amino acid sequence contained a Lys residue at the putative reactive site (position 64). ILTI was stable over a wide range of temperature and pH and in the presence of DTT.
Most intracellular parasites employ sophisticated mechanisms to direct biogenesis of a vacuolar replicative niche that circumvents default maturation through the endolysosomal cascade. However, this is not the case of the Q fever bacterium, Coxiella burnetii. This hardy, obligate intracellular pathogen has evolved to not only survive, but to thrive, in the harshest of intracellular compartments: the phagolysosome. Following internalization, the nascent Coxiella phagosome ultimately develops into a large and spacious parasitophorous vacuole (PV) that acquires lysosomal characteristics such as acidic pH, acid hydrolases and cationic peptides, defences designed to rid the host of intruders. However, transit of Coxiella to this environment is initially stalled, a process that is apparently modulated by interactions with the autophagic pathway. Coxiella actively participates in biogenesis of its PV by synthesizing proteins that mediate phagosome stalling, autophagic interactions, and development and maintenance of the mature vacuole. Among the potential mechanisms mediating these processes is deployment of a type IV secretion system to deliver effector proteins to the host cytosol. Here we summarize our current understanding of the cellular events that occur during parasitism of host cells by Coxiella.
Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short- (1 injection) and long-term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short-term and one long-term MPE treatment. One group received EV alone. Two test groups received EV with either a short-term or long-term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2-DE/MALDI-TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV-induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.
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