RESUMEN
OBJECTIVES: Since December 2016, Brazil has experienced an unusually large outbreak of yellow fever (YF). Whether urban transmission may contribute to the extent of the outbreak is unclear. The objective of this study was to characterize YF virus (YFV) genomes and to identify spatial patterns to determine the distribution and origin of YF cases in Minas Gerais, Espírito Santo and Rio de Janeiro, the most affected Brazilian states during the current YFV outbreak. METHODS: We characterized near-complete YFV genomes from 14 human cases and two nonhuman primates (NHP), sampled from February to April 2017, retrieved epidemiologic data of cases and used a geographic information system to investigate the geospatial spread of YFV. RESULTS: All YFV strains were closely related. On the basis of signature mutations, we identified two cocirculating YFV clusters. One was restricted to the hinterland of Espírito Santo state, and another formed a coastal cluster encompassing several hundred kilometers. Both clusters comprised strains from humans living in rural areas and NHP. Another NHP lineage clustered in a basal relationship. No signs of adaptation of YFV strains to human hosts were detected. CONCLUSIONS: Our data suggest sylvatic transmission during the current outbreak. Additionally, cocirculation of two distinct YFV clades occurring in humans and NHP suggests the existence of multiple sylvatic transmission cycles. Increased detection of YFV might be facilitated by raised awareness for arbovirus-mediated disease after Zika and chikungunya virus outbreaks. Further surveillance is required, as reemergence of YFV from NHPs might continue and facilitate the appearance of urban transmission cycles.
Asunto(s)
Brotes de Enfermedades , Mutación , Enfermedades de los Primates/virología , Fiebre Amarilla/epidemiología , Virus de la Fiebre Amarilla/genética , Adolescente , Adulto , Anciano , Animales , Brasil/epidemiología , Niño , Preescolar , Evolución Molecular , Femenino , Genotipo , Humanos , Lactante , Masculino , Persona de Mediana Edad , Filogenia , Primates , Fiebre Amarilla/veterinaria , Fiebre Amarilla/virología , Adulto JovenRESUMEN
We propose a probabilistic algorithm to solve the multiple sequence alignment problem. The algorithm is a simulated annealing that exploits the representation of the multiple alignment between D sequences as a directed polymer in D dimensions. Within this representation we can easily track the evolution of the alignment through local moves of low computational cost. In contrast with other probabilistic algorithms proposed to solve this problem, our approach allows the creation and deletion of gaps without extra computational cost. The algorithm was tested by aligning proteins from the kinase family. When D=3 the results are consistent with those obtained using a complete algorithm. For D>3 where the complete algorithm fails, we show that our algorithm still converges to reasonable alignments. We also study the space of solutions obtained and show that depending on the number of sequences aligned the solutions are organized in different ways, suggesting a possible source of errors for progressive algorithms. Finally, we test our algorithm in artificially generated sequences and prove that it may perform better than progressive algorithms. Moreover, in those cases in which a progressive algorithm works better, its solution may be taken as an initial condition of our algorithm and, again, we obtain alignments with lower scores and more relevant from the biological point of view.
Asunto(s)
Algoritmos , Biopolímeros/química , Proteínas/química , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Datos de Secuencia MolecularRESUMEN
Coprecipitation and cocrystallization of proteins with synthetic dyes are known to involve reversible denaturation processes which can offer specificity towards a target protein. Although the knowledge of conformational equilibrium and how to control it are central into the basic molecular dynamics of protein precipitation, the exact molecular mechanism of the precipitation remains unknown. Aiming at understanding the events that take place before the coprecipitation step of generic dye-protein systems, we investigated the binding of flavianic acid to bovine trypsin, using approaches of visible and second-derivative ultraviolet spectroscopy, viscosimetry, densimetry and circular dichroism. The results suggest a restricted transconformation of the macromolecule linked to dye binding at a stoichiometry of 1:1. An increase on the protein secondary structures occurred together with an electro-constriction effect on trypsin, a borderline event to the coprecipitation process, suggesting a stabilized structure for trypsin as a ligand-induced molten globule-like state.
Asunto(s)
Colorantes/metabolismo , Ácidos Sulfónicos/metabolismo , Tripsina/química , Tripsina/metabolismo , Ácidos/metabolismo , Dicroismo Circular , Colorantes/química , Cristalización , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Solubilidad , Espectrofotometría Ultravioleta , Ácidos Sulfónicos/química , Volumetría , ViscosidadRESUMEN
Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule
Asunto(s)
Animales , Bovinos , Colorantes/química , Proteínas/análisis , Tripsina/química , Precipitación Química , Colorantes/metabolismo , Diálisis , Modelos Teóricos , Factores de Tiempo , Tripsina/metabolismoRESUMEN
Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.
Asunto(s)
Colorantes/química , Proteínas/análisis , Ácidos Sulfónicos/química , Tripsina/química , Animales , Bovinos , Precipitación Química , Diálisis , Modelos Químicos , Factores de TiempoRESUMEN
In the 70's, pancreatic islet transplantation arose as an attractive alternative to restore normoglycemia; however, the scarcity of donors and difficulties with allotransplants, even under immunosuppressive treatment, greatly hampered the use of this alternative. Several materials and devices have been developed to circumvent the problem of islet rejection by the recipient, but, so far, none has proved to be totally effective. A major barrier to transpose is the highly organized islet architecture and its physical and chemical setting in the pancreatic parenchyma. In order to tackle this problem, we assembled a multidisciplinary team that has been working towards setting up the Human Pancreatic Islets Unit at the Chemistry Institute of the University of São Paulo, to collect and process pancreas from human donors, upon consent, in order to produce purified, viable and functional islets to be used in transplants. Collaboration with the private enterprise has allowed access to the latest developed biomaterials for islet encapsulation and immunoisolation. Reasoning that the natural islet microenvironment should be mimicked for optimum viability and function, we set out to isolate extracellular matrix components from human pancreas, not only for analytical purposes, but also to be used as supplementary components of encapsulating materials. A protocol was designed to routinely culture different pancreatic tissues (islets, parenchyma and ducts) in the presence of several pancreatic extracellular matrix components and peptide growth factors to enrich the beta cell population in vitro before transplantation into patients. In addition to representing a therapeutic promise, this initiative is an example of productive partnership between the medical and scientific sectors of the university and private enterprises.
Asunto(s)
Humanos , Ingeniería Biomédica/métodos , Diabetes Mellitus/cirugía , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/fisiología , Materiales Biocompatibles , Cápsulas , Técnicas de Cultivo/métodos , Diabetes Mellitus Tipo 1/cirugía , Matriz Extracelular , Supervivencia de Injerto , Islotes Pancreáticos/inmunologíaRESUMEN
In the 70's, pancreatic islet transplantation arose as an attractive alternative to restore normoglycemia; however, the scarcity of donors and difficulties with allotransplants, even under immunosuppressive treatment, greatly hampered the use of this alternative. Several materials and devices have been developed to circumvent the problem of islet rejection by the recipient, but, so far, none has proved to be totally effective. A major barrier to transpose is the highly organized islet architecture and its physical and chemical setting in the pancreatic parenchyma. In order to tackle this problem, we assembled a multidisciplinary team that has been working towards setting up the Human Pancreatic Islets Unit at the Chemistry Institute of the University of São Paulo, to collect and process pancreas from human donors, upon consent, in order to produce purified, viable and functional islets to be used in transplants. Collaboration with the private enterprise has allowed access to the latest developed biomaterials for islet encapsulation and immunoisolation. Reasoning that the natural islet microenvironment should be mimicked for optimum viability and function, we set out to isolate extracellular matrix components from human pancreas, not only for analytical purposes, but also to be used as supplementary components of encapsulating materials. A protocol was designed to routinely culture different pancreatic tissues (islets, parenchyma and ducts) in the presence of several pancreatic extracellular matrix components and peptide growth factors to enrich the beta cell population in vitro before transplantation into patients. In addition to representing a therapeutic promise, this initiative is an example of productive partnership between the medical and scientific sectors of the university and private enterprises.
Asunto(s)
Ingeniería Biomédica/métodos , Diabetes Mellitus/cirugía , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/fisiología , Materiales Biocompatibles , Cápsulas , Técnicas de Cultivo/métodos , Diabetes Mellitus Tipo 1/cirugía , Matriz Extracelular , Supervivencia de Injerto , Humanos , Islotes Pancreáticos/inmunologíaRESUMEN
A possible anti-inflammatory effect of intraduodenally administered trypsin was investigated using the paw oedema and pleurisy models of carrageenan-induced inflammation in rats. No anti-inflammatory effect was detected by plethysmography or on the basis of pleural leukocyte migration in treated animals compared to a sham group. The groups were implanted with an intraduodenal catheter after treatment with metopyrone, an inhibitor of endogenous corticosteroid synthesis. Since surgical stress induces an anti-inflammatory effect of its own; the sham group was an important control. Metopyrone antagonized surgical stress, and trypsin inhibited oedema by about 16% four hours after carrageenan administration, a nonsignificant reduction. Evans blue dye protein leakage into the peritoneal cavity as a measure of vascular permeability demonstrated a pro-inflammatory effect of trypsin. The present results lead us to propose that trypsin may be acting not as anti-inflammatory agent but by accelerating the inflammatory process, thereby reducing the duration of the process.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Mediadores de Inflamación/farmacología , Tripsina/farmacología , Animales , Permeabilidad Capilar , Pletismografía , RatasRESUMEN
Screening for digestive glycosidases in different parts of the gut and associated organs of Lutzomyia longipalpis is reported. Searches for the enzymes were made in blood-fed and non-blood-fed females and the enzymes were characterized as soluble or membrane-bound molecules. A total of four different activities were detected, corresponding to the following specificities: an alpha-glucosidase, an N-acetyl-beta-d-glucosaminidase, an N-acetyl-beta-d-galactosaminidase, and an alpha-l-fucosidase. Their possible role and importance for Leishmania development are discussed and the alpha-glucosidase enzyme was partially characterized. The pH inside the gut of non-blood-fed phlebotomines was measured with pH indicator dyes. The pH ranges obtained for crop, midgut, and hindgut were, respectively, higher than pH 6, pH 6, and lower than pH 6. A hypothesis concerning these data and Leishmania development is proposed.
Asunto(s)
Glicósido Hidrolasas/análisis , Insectos Vectores/enzimología , Leishmania/crecimiento & desarrollo , Psychodidae/enzimología , Animales , Metabolismo de los Hidratos de Carbono , Digestión , Femenino , Glicósido Hidrolasas/química , Concentración de Iones de Hidrógeno , Insectos Vectores/química , Insectos Vectores/parasitología , Nitrofenoles/química , Psychodidae/química , Psychodidae/parasitología , Solubilidad , Especificidad por SustratoRESUMEN
Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme
Asunto(s)
Aminoácidos/química , Colorantes/análisis , Proteínas/análisis , Inhibidores de Tripsina/química , Precipitación Química , Cinética , Modelos Químicos , Espectrofotometría , Inhibidores de Tripsina/aislamiento & purificación , Tripsina/aislamiento & purificaciónRESUMEN
A soluble proteinase was purified 90-fold from extracts of promastigotes of Leishmania (Leishmania) amazonensis using a combination of ion-exchange chromatography in Q-Sepharose Fast Flow, gel filtration chromatography in Sephacryl HR S-200, and chromatofocusing. The enzyme appeared as a single band with an apparent molecular weight of 101 kDa by silver staining following SDS-PAGE, under both reducing and nonreducing conditions. The proteinase has a pH optimum between 8.0 and 8.5 and an isoelectric point between 5.12 and 5.23, belongs to the serine proteinase class, and is inhibited by Mg2+, Ca2+, and K+. The primary specificity determined using synthetic substrates is for basic amino acids. The kinetic parameters for the Bz-L-Arg-Nam substrate are Km = 26 microM, kcat = 32 min(-1), and Ksi = 1270 microM (the proteinase showed inhibition by excess substrate). The enzyme does not hydrolyze casein, albumin, and gelatin or large peptides like the oxidized insulin B chain, but hydrolyzes small peptides like bradykinin and fragment 4-10 of the adrenocorticotropic hormone, at the carboxyl side of basic residues and aromatic residues preceding basic residues. The enzyme appears, thus, to be restricted in its action, cleaving only small peptide substrates, which characterizes the proteinase as an oligopeptidase. This is the first report of purification of a serine peptidase from Leishmania species and it increases the short list of known oligopeptidases.
Asunto(s)
Leishmania mexicana/enzimología , Serina Endopeptidasas/aislamiento & purificación , Animales , Calcio/farmacología , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Punto Isoeléctrico , Cinética , Magnesio/farmacología , Potasio/farmacología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Especificidad por SustratoRESUMEN
Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine beta-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using alpha-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of beta-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 microM; kinetic parameters for the substrate hydrolysis were: Km = 32 microM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.
Asunto(s)
Aminoácidos/química , Colorantes/análisis , Inhibidores de Tripsina/química , Precipitación Química , Concentración de Iones de Hidrógeno , Industria TextilRESUMEN
A serine protease enzyme was purified from Lachesis muta muta venom, with 40% yield, by gel filtration on Sephadex G-100 and affinity chromatography on Sepharose-agmatin. Homogeneity of the enzyme preparation was demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the enzyme had a relative mol. wt of 45,000. The molar extinction coefficient at 280 nm was 62,127 (M x cm)-1. The enzyme hydrolysed Bz-Arg-Nan with Ks = 0.233 +/- 0.08 mM and kcat = 2.80 +/- 0.07 sec-1. All the amidines and guanidines tested for their inhibitory effect on thrombin-like enzyme behaved as competitive inhibitors of the enzyme with Ki values in the range 6.2 microM to 42.3 mM for amidines and 0.19 mM to 9.31 mM for guanidines. Dissociation constant values were analyzed in terms of the binding of the inhibitors with the subsite S1, the specificity pocket of the enzyme, Ki values were discussed in accordance with those for trypsin inhibition. beta-Naphthamidine was the strongest inhibitor, while guanidine was the weakest. The differences among the Ki values were interpreted in terms of the shape of the enzyme active site. For meta- and para-substituted benzamidinium ions a good correlation was found between log l/Ki and sigma Hammett values of the substituents. The substituent effects in the pi-electrons of the benzamidine ring were considered in the frame of Hückel molecular orbital theory. A model for the binding of p-benzamidine derivatives with the primary specificity S1 subsite of the enzyme active site was proposed.
Asunto(s)
Venenos de Crotálidos/enzimología , Trombina/aislamiento & purificación , Viperidae , Animales , Benzamidinas/farmacología , Sitios de Unión , Unión Competitiva , Venenos de Crotálidos/antagonistas & inhibidores , Guanidina/farmacología , Inhibidores de Serina Proteinasa/farmacología , Trombina/antagonistas & inhibidoresRESUMEN
Culture forms of Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8) produce an extracellular enzyme that hydrolyzes sucrose molecules into their component monosaccharides. This is important because phlebotomine sand flies, the invertebrate hosts of Leishmania, ingest plant sap or aphid and coccid honeydew rich in sucrose between blood meals and Leishmania promastigotes cannot uptake sucrose. The sucrase was purified and characterized; its molecular weight, estimated by gel filtration chromatography and SDS-PAGE electrophoresis, was about 73 kDa. K(m) and V(max) measured with sucrose as substrate were respectively 4.4 mM and 6.9 mumole glucose.min-1 (mg sucrase)-1, with maximum pH activity at pH 5.5. A series of natural and p-nitrophenyl-derived substrates were assayed, characterizing the enzyme as a highly specific beta-D-fructofuranoside fructohydrolase. When 11 species of Leishmania and 7 genera of trypanosomatids were screened, only the species of the genus Trypanosoma did not produce an enzyme with saccharolytic activity. These data are in agreement with the fact that the latter vectors do not acquire sucrose or raffinose in their meals. Searching for glycolytic enzymes other than sucrase, we found an N-acetyl-beta-D-galactosaminolytic activity. This N-acetyl-galactosaminidase, here described for the first time, might have a role in peritrophic membrane disruption. The importance of sucrase and N-acetyl-beta-D-galactosaminidase in the Leishmania life cycle is discussed.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Insectos Vectores/parasitología , Leishmania mexicana/enzimología , Psychodidae/parasitología , Sacarasa/aislamiento & purificación , Animales , Metabolismo de los Hidratos de Carbono , Secuencia de Carbohidratos , Carbohidratos/química , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Medios de Cultivo , Leishmania mexicana/crecimiento & desarrollo , Datos de Secuencia Molecular , Sacarasa/metabolismo , Sacarosa/química , Sacarosa/metabolismoRESUMEN
Gut absorption is one of the first requirements for the study of the mechanism of a possible anti-inflammatory action of proteases, such as orally administered trypsin. Porcine trypsin absorption was studied in isolated jejunal loops of rats (female Holtzman and male Wistar) and guinea pigs (males) by open-loop perfusion. Trypsin was dissolved in Tyrode solution and the solution perfused at a rate of 0.5 ml/min, at 37 degrees C. Trypsin activity, total protein, and sodium and potassium concentrations were assayed in the jejunal effluent; the values were unchanged throughout the experiments, which lasted 45 to 120 min. Using a high sensitivity ELISA (i.e. pg/ml), trypsin absorption could be demonstrated by determination of the enzyme in the mesenteric venous blood (samples of 0.5 ml); the enzyme concentration increased with time of perfusion. The linear range-specificity for intact trypsin varied from 1 to 500 ng/well. In this assay polyclonal antibodies prepared against trypsin-TLCK were utilized. Whereas trypsin concentration in the perfused lumen was practically constant at 0.12 mg/ml, the concentration of absorbed trypsin in mesenteric vein blood increased from about 100 ng/ml at time zero to 1.8 micrograms/ml, after 45 min of perfusion. Histological and ultrastructural examination of the jejunal mucosa before and after perfusion revealed that the brush-border, basal membrane, and junctional complexes were fully preserved, thus eliminating the possibility that trypsin might have destroyed the structures, thereby reaching the blood circulation. The present data indicate that micrograms quantities of trypsin were absorbed by the isolated jejunal loop of the rat.
Asunto(s)
Absorción Intestinal , Yeyuno/metabolismo , Tripsina/metabolismo , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Cobayas , Yeyuno/ultraestructura , Masculino , Perfusión/métodos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Tripsina/análisisRESUMEN
Gut absorption is one of the first requirements for the study of the mechanism of a possible anti-inflammatory action of proteases, such as orally administered trypsin. Porcine trypsin absorption was studied in isolated jejunal loops of rats (female Holtzman and male Wistar) and guinea pig (males) by open-loop perfusion. Trypsin was dissolved in Tyrode solution and the solution perfused at a rate of 0.5 ml/min, at 37§C. Trypsin activity, total protein, and sodium and potassium concentrations were assayed in the jejunal effluent; the values were unchanged throughout the experiments, which lasted 45 to 120 min. Using a high sensitivity ELISA (i.e. pg/ml), trypsin absorption could be demonstrated by determination of the enzyme in the mesenteric venous blood (samples of 0.5 ml); the enzyme concentration increased with time of perfusion. The linear range-specificity for intact trypsin varied from 1 to 500 ng/well. In this assay polyclonal antibodies prepared against trypsin-TLCK were utilized. Whereas trypsin concentration in the perfused lumen was practically constant at 0.12 mg/ml, the concentration of absorbed trypsin in mesenteric vein blood increased from about 100 ng/ml at time zero to 1.8 µg/ml, after 45 min of perfusion. Histological and ultrastructural examination of the jejunal mucosa before and after perfusion revealed that the brush-border, basal membrane, and junctional complexes were fully preserved, thus eliminating the possibility that trypsin might have destroyed the structures, thereby reaching the blood circulation. The present data indicate that µg quantities of trypsin were absorbed by the isolated jejunal loop of the rat
Asunto(s)
Animales , Masculino , Femenino , Ratas , Cobayas , Absorción Intestinal , Yeyuno/metabolismo , Tripsina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Yeyuno/ultraestructura , Perfusión/métodos , Ratas Sprague-Dawley , Ratas Wistar , Tripsina/análisisRESUMEN
Identification of the substrate activation site of beta-trypsin by a 1:1 reaction with p-diazoniumbenzamidine chloride was confirmed by spectral analysis. Proteolysis of Cm-p-benzamidino-azo-beta-trypsin provided peptides containing modified tyrosine residues. The major product, Ser-146 to Lys-156, which corresponded to labeling at Tyr-151, was recovered in 35% yield, and its structure was demonstrated by amino acid analysis, Edman degradation, and mass spectrometry. Yields of labeled Tyr-151, Tyr-39, and Tyr-172, identified by peptide analysis, were in the proportion of 100:7:3. Tyr-151-(p-benzamidino)-azo-beta-trypsin is permanently activated, but can be further activated by substrates. Values of kcat, Ks', and kcat' vary from two to three times the equivalent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitor of beta-trypsin, was a hyperbolic competitive inhibitor of azo-beta-trypsin. Thus, Tyr-151, part of subsite S'2, affects the catalytic process and, when modified covalently, permanently activates trypsin. Equilibrium binding with berenil supported the kinetic data obtained with substrates. This permits the integration of protein modification, kinetics, equilibrium binding, and crystallographic data to demonstrate a fine interaction between subsites S1-S3 and S'2 in trypsin and azo-beta-trypsin, resulting in subtle structural changes when the native enzyme is covalently modified at Tyr-151.
Asunto(s)
Tripsina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Benzamidinas , Sitios de Unión , Bovinos , Compuestos de Diazonio , Activación Enzimática , Cinética , Datos de Secuencia Molecular , Tripsina/química , Inhibidores de Tripsina/farmacologíaRESUMEN
1. Competitive parabolic inhibition, a rare type of inhibition of one-substrate enzymes, is described for alpha- and beta-trypsin. The enzymes were so inhibited by two bis-benzamidines, 4-4'-diazoamino-bis-benzamidine, Berenil (DABB) and its platinum complex, DABB-PtCl2, acting on acyl-amino acid and -peptidyl nitroanilides (Nan) substrates, when inhibitor concentrations exceed 10 mM and approach the millimolar range. 2. The type of nonlinear inhibition observed requires ternary complex formation between one enzyme molecule and two inhibitor molecules (M.E.M), and also permits the formation of the mixed ternary complex (M.E.S). Binding of the first DABB molecule to the active center of trypsin takes place with Ki values of ca. 1.50 microM for both alpha- and beta-trypsin. The secondary binding site binds the inhibitor with dissociation constants Ki2 close to 0.25 mM for both forms of the enzyme, as determined with different substrates. 3. The dissociation constants of the ternary mixed complexes (Ksi and Kis), however, depend on the structural features of the substrates, which are of negligible importance for Bz-Arg-Nan, but significant for Ac-Phe-Arg-Nan and D-Val-Leu-Arg-Nan, reflecting subsite interactions between S1-S3 and S'2. 4. Pentamidine, a diamidino-4,4'-diphenoxy-alkane with a flexible chain, behaved as a strict competitive inhibitor. This implies that the triazene moiety of DABB is involved in the interaction between the inhibitor and the secondary binding site of the enzyme.
Asunto(s)
Diminazeno/análogos & derivados , Diminazeno/farmacología , Inhibidores de Tripsina/farmacología , Tripsina/metabolismo , Animales , Bovinos , Técnicas In Vitro , Cinética , Compuestos Organoplatinos/farmacología , Pentamidina/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Competitive parabolic inhibition, a rare type of inhibition of one-substrate enzymes, is described for alpha-and beta-trypsin. The enzymes were so inhibited by two bis-benzamidines 4-4' diazoamino-bis-benzamidine, Berenil (DABB) and its platinum complex, DABB-PtCl2, acting on acyl-amino acid-peptidyl nitroanilides (Nan) substrates, when inhibitor concentrations exceed 10 mM and approch the millimolar range. The type of nonlinear inhibition observed require4s ternary complex formation between one enzyme molecule and two inhibitor molecules (M. E. M), and also permits the formation of the mixed ternary complex (M. E. S.). Binding of the first DABB molecule to the active center of trypsin takes place with K1 values of ca. 1.50 uM for both alpha-and- beta-trypsin. The secondary binding site binds the inhibitor with dissociation constants K12 close to 0.25 mM for both forms of the enzyme, as determined with different substrates. The dissociation constants of the ternary mixed complexes (Ksi and Kis), however, depend on the structural features of the substrates, which are of negligible importance for Bz-Arg-Nan, but significant for Ac-Phe-Arg-Nan and D-Val-Leu-Arg-Nan, reflecting subsite interactions between S1-S3 and S'2. Pentamidine, a diamidino-4,4'-diphenoxy-alkane with a flexible chain, behaved as a strict competitive inhibitor. This implies that the triazene moiety of DABB is involved in the interaction between the inhibitor and the secondary binding site of the enzyme
Asunto(s)
Benzamidinas , Bovinos , Inhibidores Enzimáticos , Tripsina/antagonistas & inhibidoresRESUMEN
The reactivity of mononuclear cells (2 x 10**6/ml minimum Eagle's medium, MEM) from normal subjects and from Schistosoma mansoni-infected patients was evaluated by microcalorimetry. The results which are reported as heat production (mcal for 2 x 10**6 cells in 3600s), were 2,087 ñ 21.2 and 2,497.0 ñ 21.3 for mononuclear cells from infected patients (N = 8) under stimulation with S. mansoni soluble egg antigen (SEA) and soluble adult worm antigenic preparation (SWAP), respectively. The values for cells from normal subjects (N = 8) were 13.7 ñ 1.1 and 29.3 ñ 3.2 in the presence of the same antigens. Pre-treatment of mononuclear cells from patients with 1 mM aminophylline (a cAMP phospphodiesterase inhibitor) totally abolished heat production. Cell viability (> 95%) was not changed after the measurement. The microcalorimetric assay described here measures the cellular metabolic activity and we feed justified in suggesting this techinique as an auxiliary diagnosis of schistosomiasis. Given the sensitivity, precision and accuracy of this microcalorimetric assay, we feel it can be used for the diagnosis of disease conditions for which a reliable diagnostic method is required