RESUMEN

The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudo-aspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it's non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it's possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries.

Asunto(s)

Proteínas de Artrópodos/metabolismo , Proteasas de Ácido Aspártico/metabolismo , Sistema Digestivo/enzimología , Rhipicephalus/enzimología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/aislamiento & purificación , Proteasas de Ácido Aspártico/genética , Proteasas de Ácido Aspártico/aislamiento & purificación , Bovinos/parasitología , Clonación Molecular , Femenino , Regulación Enzimológica de la Expresión Génica , Seudogenes/genética , Interferencia de ARN , Rhipicephalus/genética , Rhipicephalus/fisiología , Homología de Secuencia de Aminoácido , Infestaciones por Garrapatas/parasitología

RESUMEN

Blood coagulation is an important process in haemostasis, and disorders of blood coagulation can lead to an increased risk of haemorrhage and thrombosis. Coagulation is highly conserved in mammals and has been comprehensively studied in humans in the investigation of bleeding or thrombotic diseases. Some substances can act as inhibitors of blood coagulation and may affect one or multiple enzymes throughout the process. A specific thrombin inhibitor called infestin has been isolated from the midgut of the haematophagous insect Triatoma infestans. Infestin is a member of the nonclassical Kazal-type serine protease inhibitors and is composed of four domains, all of which have a short central α-helix and a small antiparallel ß-sheet. Domains 1 and 4 of infestin (infestins 1 and 4) possess specific inhibitory activities. Infestin 1 inhibits thrombin, while infestin 4 is an inhibitor of factor XIIa, plasmin and factor Xa. Here, the structure determination and structural analysis of infestin 1 complexed with trypsin and of infestin 4 alone are reported. Through molecular modelling and docking, it is suggested that the protein-protein binding site is conserved in the infestin 1-thrombin complex compared with other Kazal-type inhibitors. Infestin 4 is able to bind factor XIIa, and the F9N and N11R mutants selected by phage display were shown to be more selective for factor XIIa in comparison to the wild type.

Asunto(s)

Proteínas de Insectos/química , Triatoma/química , Animales , Proteínas de Insectos/metabolismo , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Homología Estructural de Proteína , Trombina/química , Trombina/metabolismo

RESUMEN

Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor X(a). Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals.

Asunto(s)

Fibrinolíticos/farmacología , Trombina/antagonistas & inhibidores , Animales , Coagulación Sanguínea/efectos de los fármacos , Fibrinolíticos/aislamiento & purificación , Humanos , Trombina/química

RESUMEN

A novel method of antithrombin (AT) purification from Bothrops jararaca snake plasma was developed to obtain this protein using a waste supernatant from B. jararaca fibrinogen purification. The AT purification was achieved by affinity chromatography on HiTrap Heparin HP. The results showed an efficient purification process yielding pure AT (purity 65-fold and specific activity 368.91). In conclusion, we showed a feasible purification method of AT from B. jararaca plasma using a discarded material. This feature is important, considering the limitation of material, such as snake plasma, and could also be useful to obtain pure plasma proteins from other animals, including human plasma.

Asunto(s)

Antitrombinas/aislamiento & purificación , Bothrops/metabolismo , Fibrinógeno/aislamiento & purificación , Adsorción , Sulfato de Amonio/química , Animales , Antitrombinas/química , Compuestos de Bario/química , Bioquímica/métodos , Biomarcadores/metabolismo , Coagulación Sanguínea , Bovinos , Cloruros/química , Cromatografía de Afinidad/métodos , Electroforesis en Gel de Poliacrilamida , Fibrinógeno/química , Trombina/química

RESUMEN

A novel method of antithrombin (AT) purification from Bothrops jararaca snake plasma was developed to obtain this protein using a waste supernatant from B. jararaca fibrinogen purification. The AT purification was achieved by affinity chromatography on HiTrap Heparin HP. The results showed an efficient purification process yielding pure AT (purity 65-fold and specific activity 368.91). In conclusion, we showed a feasible purification method of AT from B. jararaca plasma using a discarded material. This feature is important, considering the limitation of material, such as snake plasma, and could also be useful to obtain pure plasma proteins from other animals, including human plasma.

Asunto(s)

Animales , Antitrombinas/aislamiento & purificación , Bothrops , Serpientes/clasificación , Venenos de Serpiente/uso terapéutico , Coagulación Sanguínea , Cromatografía

RESUMEN

Bowman-Birk inhibitors (BBIs) are cysteine-rich and highly cross-linked small proteins that function as specific pseudosubstrates for digestive proteinases. They typically display a "double-headed" structure containing an independent proteinase-binding loop that can bind and inhibit trypsin, chymotrypsin and elastase. In the present study, we used computational biology to study the structural characteristics and dynamics of the inhibition mechanism of the small BBI loop expressing a 35-amino acid polypeptide (ChyTB2 inhibitor) which has coding region for the mutated chymotrypsin-inhibitory site of the soybean BBI. We found that in the BBI-trypsin inhibition complex, the most important interactions are salt bridges and hydrogen bonds, whereas in the BBI-chymotrypsin inhibition complex, the most important interactions are hydrophobic. At the same time, ChyTB2 mutant structure maintained the individual functional domain structure and excellent binding/inhibiting capacities for trypsin and chymotrypsin at the same time. These results were confirmed by enzyme-linked immunosorbend assay experiments. The results showed that modeling combined with molecular dynamics is an efficient method to describe, predict and then obtain new proteinase inhibitors. For such study, however, it is necessary to start from the sequence and structure of the mutant interacting relatively strongly with both trypsin and chymotrypsin for designing the small BBI-type inhibitor against proteinases.

Asunto(s)

Endopeptidasas/metabolismo , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Quimotripsina/antagonistas & inhibidores , Análisis por Conglomerados , Diseño de Fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Programas Informáticos , Propiedades de Superficie , Inhibidores de Tripsina/química

RESUMEN

Every hematophagous invertebrate studied to date produces at least one inhibitor of coagulation. Among these, thrombin inhibitors have most frequently been isolated. In order to study the thrombin inhibitor from Triatoma brasiliensis and its biological significance for the bug, we sequenced the corresponding gene and evaluated its biological function. The T. brasiliensis intestinal thrombin inhibitor, termed brasiliensin, was sequenced and primers were designed to synthesize double strand RNA (dsRNA). Gene knockdown (RNAi) was induced by two injections of 15mug of dsRNA into fourth instar nymphs. Forty-eight hours after the second injection, bugs from each group were allowed to feed on hamsters. PCR results showed that injections of dsRNA reduced brasiliensin expression in the anterior midgut by approximately 71% in knockdown nymphs when compared with controls. The reduction in gene expression was confirmed by the thrombin inhibitory activity assay and the citrated plasma coagulation time assay which showed activity reductions of approximately 18- and approximately 3.5-fold, respectively. Knockdown nymphs ingested approximately 39% less blood than controls. In order to confirm the importance of brasiliensin in blood ingestion, fourth instar nymphs were allowed to ingest feeding solution alone or feeding solution containing 15U of thrombin prior to blood feeding. Fifty-five percent less blood was ingested by nymphs which were fed thrombin prior to blood feeding. The results suggest that anticoagulant activity in the midgut is an important determinant of the amount of blood taken from the host. The role of anticoagulants during blood ingestion is discussed in the light of this novel insight.

Asunto(s)

Anticoagulantes/aislamiento & purificación , Cricetinae/parasitología , Conducta Alimentaria/fisiología , Interferencia de ARN/fisiología , Trombina/antagonistas & inhibidores , Triatoma/fisiología , Animales , Tracto Gastrointestinal/química , Proteínas de Insectos/farmacología , Análisis de Secuencia/métodos , Trombina/metabolismo

RESUMEN

Bowman-Birk inhibitors (BBIs) are cysteine-rich and highly cross-linked small proteins that function as specific pseudosubstrates for digestive proteinases. They typically display a "double-headed" structure containing an independent proteinase-binding loop that can bind and inhibit trypsin, chymotrypsin and elastase. In the present study, we used computational biology to study the structural characteristics and dynamics of the inhibition mechanism of the small BBI loop expressing a 35-amino acid polypeptide (ChyTB2 inhibitor) which has coding region for the mutated chymotrypsin-inhibitory site of the soybean BBI. We found that in the BBI-trypsin inhibition complex, the most important interactions are salt bridges and hydrogen bonds, whereas in the BBI-chymotrypsin inhibition complex, the most important interactions are hydrophobic. At the same time, ChyTB2 mutant structure maintained the individual functional domain structure and excellent binding/inhibiting capacities for trypsin and chymotrypsin at the same time. These results were confirmed by enzyme-linked immunosorbend assay experiments. The results showed that modeling combined with molecular dynamics is an efficient method to describe, predict and then obtain new proteinase inhibitors. For such study, however, it is necessary to start from the sequence and structure of the mutant interacting relatively strongly with both trypsin and chymotrypsin for designing the small BBI-type inhibitor against proteinases.

Asunto(s)

Animales , Endopeptidasas/metabolismo , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Modelos Moleculares , Secuencia de Aminoácidos , Bovinos , Análisis por Conglomerados , Diseño de Fármacos , Inhibidor de la Tripsina de Soja de Bowman-Birk/farmacología , Inhibidores de Tripsina/química , Quimotripsina/antagonistas & inhibidores

RESUMEN

Infestin is a protein from Triatoma infestans (kissing bug) composed of seven Kazal-type domains that is further processed to yield several serine protease inhibitors with varying specificities. Infestins 3 and 4 are the last two domains of the infestin gene and are found in vivo in the insect's anterior midgut. The last domain, infestin 4, has been cloned, expressed and purified. Here, the crystallization of infestin 4 using the sitting-drop vapour-diffusion method with PEG 8000 as precipitant is described. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 25.89, b = 45.64, c = 57.41 A. X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source. Initial phases were calculated by molecular replacement using an edited rhodniin molecule as the search model. Structure refinement is in progress.

Asunto(s)

Proteínas Sanguíneas/química , Proteínas de Insectos/química , Triatoma/química , Animales , Cristalización , Cristalografía por Rayos X

RESUMEN

This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively).

Asunto(s)

Proteínas de Insectos/genética , Inhibidores de Serina Proteinasa/genética , Trombina/antagonistas & inhibidores , Triatoma/genética , Secuencia de Aminoácidos , Animales , Enfermedad de Chagas , Clonación Molecular , Sistema Digestivo , Expresión Génica , Genes de Insecto , Proteínas de Insectos/metabolismo , Insectos Vectores , Datos de Secuencia Molecular , Inhibidores de Serina Proteinasa/metabolismo

RESUMEN

The present study describes the purification, characterization, and comparison of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs between the first day of hatching and the beginning of eclosion (defined as El, E2, and E3) and of larvae between the first day of eclosion and the infectant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCI buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from E1 to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant Ki determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and larvae (BmTls) were purified on trypsin-Sepharose column and analyzed by SDS-PAGE. The results showed a slight difference in protein pattern, with a protein band of 20 kDa in the E1 and E2 samples which did not appear in the other samples. The Ki for neutrophil elastase was 10-fold lower in L3 than E1. BmTI reverse-phase chromatography showed two and one major peaks in egg and larva samples, respectively. The N-terminal amino acid sequence of the L3 main peak from a C8 column showed a mix of BmTIs with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition activity suggest different roles for BmTIs during the development process.

Asunto(s)

Inhibidores de Serina Proteinasa/aislamiento & purificación , Garrapatas/metabolismo , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Larva/metabolismo , Datos de Secuencia Molecular , Óvulo/química , Proteínas/análisis , Inhibidores de Serina Proteinasa/metabolismo , Garrapatas/embriología

RESUMEN

Salivary anticoagulant activities are widely distributed among hematophagous arthropods. Most of them are inhibitors of the serine proteases of the coagulation cascade. Here we show that the saliva of the exclusively hematophagous insect Triatoma infestans, an important vector in the transmission of Chagas' disease, contains an uncommon trypsin-like activity, triapsin. This novel enzyme was purified and characterized. It is a serine protease that is stored as a zymogen in the luminal content of the salivary glands D2. Triapsin is activated by trypsin treatment, or when the saliva is ejected during the insect bite. The enzyme was purified 300-fold from the released saliva by anion exchange chromatography in a HiTrap Q column, followed by chromatography in Phenyl-Superose, and Superdex HR75. The purified triapsin shows an apparent molecular mass of around 40 kDa in non-reduced SDS gels and in sieving chromatography, and 33 kDa in reduced SDS-gels. Its activity is lost after incubation with dithiothreitol indicating that cysteine bridges are essential for activity. Triapsin cleaves gelatin and synthetic substrates showing preference for arginine at P1 residues. The best p-nitroanilide substrate is isoleucyl-prolyl-arginine. It does not cleave bradykinin, angiotensin and other lysine containing substrates. The triapsin amidolytic activity against chromogenic substrates is similar to plasminogen activators, such as urokinase and tissue plasminogen activator. However, it does not activate plasminogen. The fact that triapsin is released at the bite in its active form suggests that it has a role in blood feeding.

Asunto(s)

Proteínas de Insectos/aislamiento & purificación , Insectos Vectores/enzimología , Saliva/enzimología , Triatoma/enzimología , Tripsina/aislamiento & purificación , Animales , Sangre , Enfermedad de Chagas/transmisión , Precursores Enzimáticos/metabolismo , Conducta Alimentaria , Mordeduras y Picaduras de Insectos , Proteínas de Insectos/metabolismo , Activadores Plasminogénicos/aislamiento & purificación , Activadores Plasminogénicos/metabolismo , Inhibidores de Proteasas/farmacología , Especificidad por Sustrato , Tripsina/metabolismo

RESUMEN

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The Km values determined for three different substrates were 1.88 x 10(-4), 1.28 x 10(-4), and 1.40 x 10(-4) M for H-alpha-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), DL-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SB

Asunto(s)

Fibrinolíticos/aislamiento & purificación , Muscidae/enzimología , Serina Endopeptidasas/aislamiento & purificación , Animales , Fibrina/metabolismo , Fibrinolíticos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Peso Molecular , Muscidae/metabolismo , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato

RESUMEN

Preying on cattle, the hard tick Boophilus microplus causes heavy economical losses to Brazil. Tick proteins are a good target to be used as tools for tick control. Serine protease inhibitors from B. microplus larvae (BmTI) were preliminarily characterized. One-week-old larvae were the source of a 2% protein solution in 5 mM Tris-HCl, 20 mM NaCl, pH 7.4. The inhibitors were purified by affinity chromatography on trypsin-Sepharose, and ion-exchange chromatography on Resource Q column, and they separated in two major active peaks, corresponding to 10-kDa and 18-kDa proteins (BmTI-B and BmTI-A, respectively). Both purified proteins inhibited trypsin with Ki of 0.3 and 3.0 nM, respectively, but only the 18-kDa protein inhibited elastase (Ki 1.4 nM) and plasma kallikrein (Ki 120 nM). BmTI-A did not change prothrombin time (PT) and thrombin time (TT), but it increased activated partial thromboplastin time (APTT) was dose-dependent. The partial amino acid sequence indicated that BmTI-A belongs to the bovine pancreatic trypsin inhibitor (BPTI)-Kunitz type inhibitor family. These inhibitors (by their properties) play a role in the feeding process of the tick. Development of antibodies against these proteins may be used to impair the normal feeding and consequently, the parasite would be no longer viable.

Asunto(s)

Calicreínas/antagonistas & inhibidores , Calicreínas/sangre , Elastasa Pancreática/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/aislamiento & purificación , Garrapatas/química , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Larva/química , Datos de Secuencia Molecular , Inhibidores de Serina Proteinasa/metabolismo

RESUMEN

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.

Asunto(s)

Bacteriófagos/química , Biblioteca de Péptidos , Proteínas/química , Proteínas/farmacocinética , Serina Endopeptidasas/química , Trombina/antagonistas & inhibidores , Secuencia de Aminoácidos , Secuencia de Bases , Quimasas , Clonación Molecular , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Insercional , Saccharomyces cerevisiae/química , Trombina/efectos de los fármacos , Triptasas

RESUMEN

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.

Asunto(s)

Fabaceae/metabolismo , Plantas Medicinales , Semillas/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/aislamiento & purificación , Inhibidores de Tripsina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Bovinos , Quimotripsina/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Hidrólisis , Técnicas In Vitro , Cinética , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Ratas/inmunología , Tripsina/metabolismo , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/farmacología , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacología

RESUMEN

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds of Torresea acreana, one of the two known species of Torresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears as M(r) 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2, Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.

Asunto(s)

Plantas/química , Semillas/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Inhibidores de Tripsina/química , Secuencia de Aminoácidos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Aminoácido , Inhibidores de Tripsina/aislamiento & purificación

RESUMEN

The action of two Bowman-Birk and several plant Kunitz-type inhibitors were studied on trypsin, chymotrypsin, plasma kallikrein and factor XII. The primary structure of some of them was completely defined. The results showed that the Bowman-Birk type inhibitors, although potent inhibitors for trypsin (Ki in the range of 1-2 nM), are not able to inhibit plasma kallikrein. Factor XII (Ki = 1.4 microM) and chymotrypsin (Ki = 5.0 nM) are inhibited by Torresea cearensis trypsin inhibitor (TcTI) but not by Dioclea glabra trypsin inhibitor (DgTI). Both inhibitors reactive site regions are highly homologous, and the amino acid residues in P1 position are the same, Lys and His; major differences are in the charge of the C-terminal portion of the molecules. The studied Kunitz-type inhibitors were all able to inhibit plasma kallikrein (Ki between 4 and 80 nM), with the exception of Schizolobium parahyba chymotrypsin inhibitor (SpCI), that is specific for chymotrypsin. All Kunitz-type inhibitors inactivate chymotrypsin, but with a dissociation constant in the range of 0.1 to 0.6 microM. Factor XIIf is inhibited with Ki in the range of 0.1 microM. Bauhinia bauhinioides trypsin inhibitor (BbTI) did not promote factor XIIf inhibition. The Kunitz-type inhibitors are a highly homologous, sharing 60% identity in the N-terminal portion of the loop containing the reactive site, and 28.6% identity in the C-terminal portion of the same loop.

Asunto(s)

Calicreínas/química , Calicreínas/efectos de los fármacos , Proteínas de Plantas/farmacología , Inhibidores de Serina Proteinasa/farmacología , Inhibidor de la Tripsina de Soja de Bowman-Birk , Secuencia de Aminoácidos , Animales , Aprotinina/farmacología , Arachis/enzimología , Quimotripsina/efectos de los fármacos , Factor XII/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Tripsina/efectos de los fármacos , Inhibidores de Tripsina/farmacología

Asunto(s)

Factor XII/efectos de los fármacos , Factor X/efectos de los fármacos , Calicreínas/efectos de los fármacos , Proteínas de Plantas/farmacología , Inhibidores de Serina Proteinasa/análisis , Inhibidores de Serina Proteinasa/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Datos de Secuencia Molecular

RESUMEN

Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, Factor XIIa and human plasma kallikrein. The inhibitors were purified from Enterolobium contortisiliquum (Mr = 23,000), Torresea cearensis (Mr = 13,000), Bauhinia bauhinioides (Mr = 20,000), Bauhinia mollis (Mr = 20,000) and Bauhinia pentandra (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein. B. bauhinioides and B. pentrandra inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa, and B. mollis inhibitor causes trypsin inactivation only. Calculated Ki values were between 10(-7) and 10(-9) M. Chymotrypsin, like trypsin, is also inhibited, but with lower affinity. The trypsin inhibitors, isolated from E. contortisiliquum, B. pentandra, B. bauhinioides and B. mollis seem to be of the Kunitz type; the inhibitor purified from T. cearensis is of the Bowman-Birk type.

Asunto(s)

Fabaceae/química , Calicreínas/metabolismo , Cininas/metabolismo , Plantas Medicinales , Inhibidores de Proteasas/farmacología , Brasil , Electroforesis en Gel de Poliacrilamida , Indicadores y Reactivos , Peso Molecular , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Tripsina/aislamiento & purificación , Inhibidores de Tripsina/farmacología