RESUMEN
Triosephosphate isomerase (TIM) is a major enzyme in the glycolytic pathway, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. Here, we report cloning, expression and purification of a catalytically active recombinant TIM of Leishmania donovani (LdTIM). The recombinant LdTIM had a pH optimum in the range of 7.2-9.0, found stable at 25°C for 30 min and K(m) and V(max) for the substrate glyceraldehyde 3-phosphate was 0.328±0.02mM and 10.05mM/min/mg, respectively. The cysteine-reactive agent methylmethane thiosulphonate (MMTS) was used as probe, in order to test its effect on enzyme activity. The MMTS induced 75% enzyme inactivation within 15 min at 250 µM concentration. The biochemical characterization of LdTIM described in this work is the essential step towards deeper understanding of its role in parasite survival. The purification of LdTIM in bioactive form provides important tools for further functional and structural studies.
Asunto(s)
Leishmania donovani/enzimología , Triosa-Fosfato Isomerasa/metabolismo , Secuencia de Aminoácidos , Western Blotting , Cromatografía en Gel , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Leishmania donovani/genética , Metilmetanosulfonato/análogos & derivados , Metilmetanosulfonato/farmacología , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/aislamiento & purificaciónAsunto(s)
Malformación de Arnold-Chiari , Artropatía Neurógena , Articulación del Codo , Siringomielia , Humanos , Articulación del Codo/diagnóstico por imagen , Siringomielia/complicaciones , Siringomielia/diagnóstico por imagen , Artropatía Neurógena/complicaciones , Artropatía Neurógena/diagnóstico por imagenRESUMEN
OBJECTIVE: To investigate the frequency association between resistin gene polymorphism with its circulating levels, metabolic risk factor and insulin resistance in adult women. DESIGN: Totally 615 subjects were enrolled for the study, 305 women were with metabolic syndrome and 310 women were without metabolic syndrome according to NCEP-ATP III criteria. Fasting circulatory level of resistin, insulin, plasma glucose and lipid profiles were estimated along with calculation of insulin resistance. Resistin 420C/G promoter region polymorphism was done by RFLP method. RESULTS: Variant genotype (CC vs CG+GG) (p<0.001: OR=2.22: 95% CI=1.60-3.10) of 420C/G resistin gene polymorphism was less frequently observed in control population. Further dividing subjects into two groups according to absence (Resistin -1) or presence (Resistin-2) of the G allele, significantly high levels of triglyceride (p<0.001), plasma glucose (p=0.012), systolic blood pressure (p<0.001), diastolic blood pressure (p<0.001), waist hip ratio (p<0.001), body mass index (p<0.001) and resistin (p<0.001), were observed in resistin-2 group. CONCLUSION: Present study shows that 420C/G polymorphism of resistin gene directly correlated to its high circulating level and metabolic risk factors, specifically markers of obesity and atherosclerosis, so it may have an important role in the development of metabolic syndrome and cardio metabolic diseases.
Asunto(s)
Alelos , Síndrome Metabólico/genética , Polimorfismo de Longitud del Fragmento de Restricción , Regiones Promotoras Genéticas , Resistina/genética , Adulto , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/inmunología , Presión Sanguínea , Femenino , Humanos , Síndrome Metabólico/sangre , Síndrome Metabólico/inmunología , Obesidad/sangre , Obesidad/genética , Obesidad/inmunología , Resistina/sangre , Resistina/inmunología , Factores de Riesgo , Triglicéridos/sangre , Triglicéridos/inmunologíaRESUMEN
The triosephosphate isomerase of Leishmania donovani (LdTIM) was expressed at high level in Escherichia coli. The TIM gene was cloned in expression vector pET-23(a) with C-terminal 6× His tag fused in frame, and expressed as a 27.6-kDa protein in E. coli as inclusion bodies. The recombinant LdTIM from E. coli lysate was solubilized in 6 M guanidine hydrochloride and purified by Ni-NTA chromatography. In the present study, the effect of bovine serum albumin on the reactivation of TIM was investigated. Furthermore, 8-anilino-1-naphthalene sulfonic acid was used to detect the structural changes induced by bovine serum albumin (BSA). Here, we conclude that BSA assists in the refolding and regain of LdTIM enzyme activity by providing framework for structure formation. This study indicates that numerous protein-protein contacts are constantly occurring inside the cell that leads to the formation of native protein.
Asunto(s)
Biotecnología/métodos , Escherichia coli/enzimología , Cuerpos de Inclusión/enzimología , Leishmania donovani/enzimología , Pliegue de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica/farmacología , Triosa-Fosfato Isomerasa , Naftalenosulfonatos de Anilina/análisis , Animales , Western Blotting , Bovinos , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Vectores Genéticos , Guanidina/química , Cuerpos de Inclusión/genética , Leishmania donovani/química , Leishmania donovani/genética , Pliegue de Proteína/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Albúmina Sérica/metabolismo , Espectrometría de Fluorescencia , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
Squalene synthase (SSN, EC 2.5.1.21), a major enzyme in the sterol biosynthetic pathway, catalyses an unusual head-to-head reductive dimerization of two molecules of farnesyl-pyrophosphate (FPP) in a two-step reaction to form squalene. FPP serves as a metabolic intermediate in the formation of sterols, dolichols, ubiquinones and farnesylated proteins. Here, we report cloning, expression and purification of a catalytically active recombinant squalene synthase of Leishmania donovani (LdSSN). The pH and temperature optima of LdSSN were 7.4 and 37°C, respectively. Biochemical studies revealed that the K(m) and V(max) for the substrate FPP were 3.8 µM and 0.59 nM min(-1) mg(-1) and for NADPH were 43.23 µM and 0.56 nM min(-1) mg(-1). LdSSN was found to be sensitive towards denaturants as manifested by a loss of enzyme activity at the concentration of 1 M urea or 0.25 M guanidine hydrochloride. Zaragozic acid A, a potent inhibitor of mammalian SSN, was also a competitive inhibitor of recombinant LdSSN, with a K(i) of 74 nM. This is the first report on the purification and characterization of full-length recombinant SSN from L. donovani. Studies on recombinant LdSSN will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis.
Asunto(s)
Clonación Molecular , Farnesil Difosfato Farnesil Transferasa/química , Farnesil Difosfato Farnesil Transferasa/genética , Expresión Génica , Leishmania donovani/enzimología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Estabilidad de Enzimas , Farnesil Difosfato Farnesil Transferasa/aislamiento & purificación , Farnesil Difosfato Farnesil Transferasa/metabolismo , Cinética , Leishmania donovani/química , Leishmania donovani/clasificación , Leishmania donovani/genética , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Alineación de SecuenciaRESUMEN
Serine hydroxymethyltransferase (SHMT) catalyzes the inter conversion of serine and tetrahydrofolate (H(4)-folate) to form glycine and 5,10-methylene H(4)-folate and generates one-carbon fragments for the synthesis of nucleotides, methionine, thymidylate, choline, etc. In spite of being an indispensable enzyme of the thymidylate cycle, SHMT in Leishmania donovani remains uncharacterized. The study of L. donovani SHMT (ldSHMT) becomes important as this gene is preferentially expressed in the amastigote stage of parasite, which resides in human macrophages. Here we report cloning, expression and purification of a catalytically active ldSHMT. The homogeneity of recombinant protein was analyzed by denaturing gel electrophoresis and protein was found to be 95% pure having yield of 1mg/l. The recombinant protein is a tetramer of 216kDa as evidenced by gel filtration chromatography and uses serine and tetrahydrofolate as substrates with Km of 1.6 and 2.4mM, respectively. Further biochemical studies revealed that pH optimum of ldSHMT is 7.8 and enzyme is thermally stable up to 45 degrees C. ldSHMT was found sensitive towards denaturants as manifested by loss of enzyme activity at the concentration of 1M urea or 0.25M guanidine hydrochloride. This is the first report of purification and characterization of recombinant SHMT from any protozoan source. Studies on recombinant ldSHMT will help in evaluating this enzyme as potential drug target.
Asunto(s)
Glicina Hidroximetiltransferasa/genética , Leishmania donovani/enzimología , Animales , Clonación Molecular , Expresión Génica , Glicina Hidroximetiltransferasa/química , Glicina Hidroximetiltransferasa/clasificación , Leishmania donovani/metabolismo , Peso Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A series of anthranilodinitrile-based biaryls were synthesized and evaluated in vitro against extracellular promastigotes and intracellular amastigotes of Leishmania donovani. Among various screened compounds, a biaryl with trifluoromethyl group 5f showed 83% inhibition against promastigotes and 70% inhibition against amastigotes of L. donovani at 8 and 20microg/mL concentrations, respectively.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania donovani/efectos de los fármacos , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Línea Celular , Evaluación Preclínica de Medicamentos , RatonesRESUMEN
Various 6-aryl-3-cyano/methoxycarbonyl-4-methylsulfanyl-2H-pyran-2-ones have been synthesized as a potential substitute of 2,4-thiazolidinedione head group to express potent PPAR-gamma transactivation response. Some of the screened compounds have shown promising PPAR-gamma agonistic activity.
Asunto(s)
Nitrilos/farmacología , PPAR gamma/efectos de los fármacos , Piranos/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Ligandos , Modelos Moleculares , Estructura Molecular , Nitrilos/síntesis química , Nitrilos/química , PPAR gamma/agonistas , Piranos/síntesis química , Piranos/química , Relación Estructura-ActividadRESUMEN
Lipase isolated from a soil isolate, Pseudomonas mendocina (PK-12CS) chemoselectively hydrolyzed the fatty ester group in presence of arbamate of compound 5-amino-2,4-dihydro-3H-1,2,4-triazole-3 ones, a class of compounds which are attractive starting materials for the synthesis of triazole annealed heterocycles. The enzymatic method provides an easy access to the synthesis of N-substituted glycine. Under optimized fermentation conditions the culture produced 3510 Lipolytic Units/mL of cell free fermentation broth in 20 h of fermentation. The purified lipase exhibited molecular mass of 80 kDa on SDS polyacrylamide gel electrophoresis. The enzyme was stable at room temperature for more than a month and expressed maximum activity at 37 degrees C and pH 8.