RESUMEN
L-Tryptophan (Trp) and some of its metabolites regulate the circadian rhythm in mammals. We aimed to investigate the effects of short-term supplementation of Trp in isocaloric meals on growth performance using the parameters of multiple blood biomarkers and free amino acids in growing pigs. A total of 32 Landrace × Yorkshire barrows with a mean body weight of 8.64 (±1.13) kg were randomly assigned to four groups and then fed with various concentrations of Trp diets daily. Our results showed that sequential supplementation of different concentrations of Trp in isocaloric meals decreased the feed:gain (F:G) ratio (P = 0.079) and plasma urea and albumin (P = 0.019), whereas the level of total protein did not. Among the essential and conditionally essential amino acids, the concentrations of histidine, isoleucine, proline, threonine, arginine, and valine in the plasma decreased (P < 0.05), whereas the concentrations of Trp, glycine, serine, and methionine increased (P < 0.01). In addition, concentrations of branched chain amino acids also significantly decreased (P = 0.004), while the rate of conversion of Trp to branched chain amino acids increased (P < 0.001). Taken together, we show that administration of a high concentration of Trp in breakfast with decreasing concentrations of Trp in lunch and dinner positively affected feed utilization and improved feed efficiency, at least in part, through the optimization of amino acid interconversions and nitrogen utilization.
Asunto(s)
Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales/efectos de los fármacos , Sus scrofa/crecimiento & desarrollo , Triptófano/farmacología , Aumento de Peso/efectos de los fármacos , Aminoácidos/metabolismo , Aminoácidos de Cadena Ramificada/sangre , Aminoácidos Esenciales/sangre , Alimentación Animal/estadística & datos numéricos , Animales , Glucemia , Nitrógeno de la Urea Sanguínea , Suplementos Dietéticos , Nitrógeno/metabolismo , Sus scrofa/metabolismo , Triptófano/metabolismoRESUMEN
Mammalian Delta(1)-pyrroline-5-carboxylate synthase (P5CS) is a bifunctional ATP- and NAD(P)H-dependent mitochondrial enzyme that catalyzes the coupled phosphorylation and reduction-conversion of L: -glutamate to P5C, a pivotal step in the biosynthesis of L: -proline, L: -ornithine and L: -arginine. Previously, we reported cloning and characterization of two P5CS transcript variants generated by exon sliding that encode two protein isoforms differing only by a two amino acid-insert at the N-terminus of the gamma-glutamyl kinase active site. The short form (P5CS.short) is highly expressed in the gut and is inhibited by ornithine. In contrast, the long form (P5CS.long) is expressed ubiquitously and is insensitive to ornithine. Interestingly, we found that all the established human cell lines we have studied expressed P5CS.long but not P5CS.short. In addition, expression of P5CS.long can be modulated by hormones: downregulation by hydrocortisone and dexamethasone and upregulation by estradiol, for example. Using a quantitative proteomic approach, we showed that P5CS.long is upregulated by p53 in p53-induced apoptosis in DLD-1 colorectal cancer cells. Functional genomic analysis confirmed that there are two p53-binding consensus sequences in the promoter region and in the intron 1 of the human P5CS gene. Interestingly, overexpression of P5CS by adenoviruses harboring P5CS.long or P5CS.short in various cell types has no effect on cell growth or survival. It would be of importance to further investigate the role of P5CS as a p53 downstream effector and how P5CS.short expression is regulated by hormones and factors of alternative splicing in cells isolated from model animals.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Ornitina-Oxo-Ácido Transaminasa/genética , Ornitina-Oxo-Ácido Transaminasa/fisiología , Secuencia de Aminoácidos , Línea Celular Tumoral , Dexametasona/metabolismo , Estradiol/metabolismo , Ácido Glutámico/química , Humanos , Hidrocortisona/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Fosforilación , Prolina/química , Estructura Terciaria de Proteína , Proteómica/métodosRESUMEN
Proline oxidase (POX), often considered a 'housekeeping enzyme' might play an important role in apoptosis. We have shown that POX generated proline-dependent reactive oxygen species (ROS), specifically superoxide radicals, and induced apoptosis through the mitochondrial (intrinsic) pathway. In our current report, we used DLD-1 colorectal cancer cells stably transfected with the POX gene under the control of a tetracycline-inducible promoter and found POX-stimulated expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), DR5 and cleavage of caspase-8. Importantly, apoptosis measured by flow cytometry was partially inhibited by Z-IETD-FMK, a specific inhibitor of caspase-8. These findings suggest that the extrinsic (death receptor) pathway also is activated by POX. Furthermore, the mechanism of this effect on the extrinsic pathway, specifically, the induction of TRAIL by POX, may be mediated by NFAT transcription factors. Additionally, POX expression also dramatically decreased phosphorylation of MEK and ERK, and the decrease was partially reversed by expression of manganese superoxide dismutase (MnSOD). Overexpression of constitutively active form of MEK, acMEK, partially blocked POX-induced apoptosis. These findings suggest the involvement of MEK/ERK signaling and further confirm the role of ROS/superoxides in POX-induced apoptosis. Combined with previously published data, we conclude that POX may induce apoptosis through both intrinsic and extrinsic pathways and is involved in nuclear factor of activated T cells (NFAT) signaling and regulation of the MEK/ERK pathway. It is suggested that, as a nutrition factor, POX may modulate apoptosis signals induced by p53 or other anti-cancer agents and enhance apoptosis in stress situations.
Asunto(s)
Apoptosis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Factores de Transcripción NFATC/metabolismo , Prolina Oxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Superóxidos/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
The p53-dependent initiation of apoptosis is accompanied by the induction of proline oxidase (POX), a mitochondrial enzyme catalyzing the conversion of proline to pyrroline-5-carboxylate with the concomitant transfer of electrons to cytochrome c. However, the contribution of increased POX activity to apoptosis, if any, remains unknown. Using Adriamycin to initiate p53-dependent apoptosis, we showed that the expression of POX is up-regulated in a time- and dose-dependent manner in a human colon cancer cell line (LoVo). In cells expressing POX, the addition of proline increases reactive oxygen species (ROS) generation in a concentration-dependent manner; glutamate, a downstream product of proline oxidation, had no effect. Induction of POX was dependent on the p53 status of the cell. In the conditionally immortalized murine colonic epithelial cell line YAMC, where the p53 phenotype can be modulated by temperature, proline oxidase expression and ROS production could only be induced when the cells were phenotypically p53-positive. To confirm that the observed ROS production was not secondary to some other effect of p53, we also conditionally expressed POX in a p53-negative colon cancer line. Again, we found a proline-dependent ROS increase with POX expression. We hypothesize that proline oxidation supports the generation of ROS by donating reducing potential to an electron transport chain altered either by p53-dependent mechanisms or by overexpression of POX.
Asunto(s)
Neoplasias del Colon/enzimología , Regulación Neoplásica de la Expresión Génica/fisiología , Prolina Oxidasa/metabolismo , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Catálisis , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Doxorrubicina/farmacología , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Prolina/farmacología , Prolina Oxidasa/biosíntesis , Prolina Oxidasa/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
delta(1)-pyrroline-5-carboxylate synthase (P5CS), a bifunctional ATP- and NADPH-dependent mitochondrial enzyme, catalyzes the reduction of glutamate to delta(1)-pyrroline-5-carboxylate, a critical step in the biosynthesis of proline, ornithine and arginine. Recently, we reported the cloning and expression of human and murine P5CS cDNAs. Previously, we showed that mammalian P5CS undergoes alternative splicing to generate two isoforms differing only by a 2 amino acid insert at the N-terminus of the gamma-glutamyl kinase active site. The short isoform has high activity in the gut, where it participates in arginine biosynthesis and is inhibited by ornithine. The long isoform, expressed in multiple tissues, is necessary for the synthesis of proline from glutamate and is insensitive to ornithine. Here, we describe a newly recognized inborn error due to the deficiency of P5CS in two siblings with progressive neurodegeneration, joint laxity, skin hyperelasticity and bilateral subcapsular cataracts. Their metabolic phenotype includes hyperammonemia, hypoornithinemia, hypocitrullinemia, hypoargininemia and hypoprolinemia. Both are homozygous for the missense mutation, R84Q, which alters a conserved residue in the P5CS gamma-glutamyl kinase domain. R84Q is not present in 194 control chromosomes and dramatically reduces the activity of both P5CS isoforms when expressed in mammalian cells. Additionally, R84Q appears to destabilize the long isoform. This is the first documented report of an inborn error of P5CS and suggests that this disorder should be considered in the differential diagnosis in patients with neurodegeneration and/or cataracts and connective tissue disease.
Asunto(s)
Arginina/sangre , Citrulina/sangre , Hiperamonemia/enzimología , Hiperamonemia/genética , Ornitina-Oxo-Ácido Transaminasa/genética , Ornitina/sangre , Prolina/sangre , Adulto , Errores Innatos del Metabolismo de los Aminoácidos/sangre , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Animales , Células CHO , Dominio Catalítico/genética , Niño , Cricetinae , Análisis Mutacional de ADN , Femenino , Fibroblastos , Francia , Humanos , Hiperamonemia/sangre , Masculino , Mutación/genética , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Linaje , Fenotipo , Prolina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , TransfecciónRESUMEN
Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 encode mitochondrial carrier family (MCF) proteins that transport ornithine across the mitochondrial inner membrane. We used their sequences to identify EST candidates that partially encode orthologous mammalian transporters. We thereby identified such a gene (ORNT1) that maps to 13q14 and whose expression, similar to that of other urea cycle (UC) components, was high in liver and varied with changes in dietary protein. ORNT1 expression restores ornithine metabolism in fibroblasts from patients with hyperammonaemia-hyperornithinaemia-homocitrullinuria (HHH) syndrome. In a survey of 11 HHH probands, we identified 3 ORNT1 mutant alleles that account for 21 of 22 possible mutant ORNT1 genes in our patients: F188delta, which is common in French-Canadian HHH patients and encodes an unstable protein; E180K, which encodes a stable, properly targeted protein that is inactive; and a 13q14 microdeletion. Our results show that ORNT1 encodes the mitochondrial ornithine transporter involved in UC function and is defective in HHH syndrome.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Amoníaco/sangre , Proteínas Portadoras/genética , Cromosomas Humanos Par 13 , Citrulina/metabolismo , Proteínas de Transporte de Membrana , Ornitina/sangre , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos , Animales , Canadá , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Mapeo Cromosómico , Femenino , Francia/etnología , Tamización de Portadores Genéticos , Humanos , Cariotipificación , Masculino , Ratones , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Datos de Secuencia Molecular , Neurospora crassa/genética , Ornitina/metabolismo , Mutación Puntual , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Piel/metabolismo , Síndrome , TransfecciónRESUMEN
Delta1-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to Delta1-pyrroline-5-carboxylate, a critical step in the de novo biosynthesis of proline and ornithine. We utilized published plant P5CS sequence to search the expressed sequence tag data base and cloned two full-length human P5CS cDNAs differing in length by 6 base pairs (bp) in the open reading frame. The short cDNA has a 2379-bp open reading frame encoding a protein of 793 residues; the long cDNA, generated by "exon sliding," a form of alternative splicing, contains an additional 6-bp insert following bp +711 of the short form resulting in inclusion of two additional amino acids in the region predicted to be the gamma-glutamyl kinase active site of P5CS. The long form predominates in all tissues examined except gut. We also isolated the corresponding long and short murine P5CS transcripts. To confirm the identity of the putative P5CS cDNAs, we expressed both human forms in gamma-glutamyl kinase- and gamma-glutamyl phosphate reductase-deficient strains of Saccharomyces cerevisiae and showed that they conferred the proline prototrophy. Additionally, we found expression of the murine putative P5CS cDNAs conferred proline prototrophy to P5CS-deficient Chinese hamster ovary cells (CHO-K1). We utilized stable CHO-K1 cell transformants to compare the biochemical characteristics of the long and short murine P5CS isoforms. We found that both confer P5CS activity and that the short isoform is inhibited by L-ornithine with a Ki of approximately 0.25 mM. Surprisingly, the long isoform is insensitive to ornithine inhibition. Thus, the two amino acid insert in the long isoform abolishes feedback inhibition of P5CS activity by L-ornithine.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , 1-Pirrolina-5-Carboxilato Deshidrogenasa , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , ADN Complementario/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Ornitina/antagonistas & inhibidores , Ornitina/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
We surveyed Delta1-pyrroline 5-carboxylate dehydrogenase genes from four patients with hyperprolinemia type II using RT-PCR amplification, genomic PCR amplification and direct sequencing. We found four mutant alleles, two with frameshift mutations [A7fs(-1) and G521fs(+1)] and two with missense mutations (S352L and P16L). To test the functional consequences of three of these, we expressed them in a P5CDh-deficient strain of Saccharomyces cerevisiae . In contrast to wild-type human P5CDh, yeast expressing S352L and G521fs(+1) failed to grow on proline and had no detectable P5CDh activity. The P16L allele, however, produced fully functional P5CDh and subsequent analysis suggests that it is polymorphic in the relevant (Spanish) population. Interestingly, the G521fs(+1) allele segregates in the large Irish Traveller pedigree used to define the HPII phenotype. To our knowledge, this is the first description of the molecular basis for this inborn error.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Prolina/metabolismo , 1-Pirrolina-5-Carboxilato Deshidrogenasa , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/clasificación , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Mutación del Sistema de Lectura , Expresión Génica , Humanos , Masculino , Linaje , Mutación Puntual , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CDh; EC 1.5.1.12), a mitochondrial matrix NAD(+)-dependent dehydrogenase, catalyzes the second step of the proline degradation pathway. Deficiency of this enzyme is associated with type II hyperprolinemia (HPII), an autosomal recessive disorder characterized by accumulation of delta 1-pyrroline-5-carboxylate (P5C) and proline. As an initial step in understanding the biochemistry of human P5CDh and molecular basis of HPII, we utilized published peptide sequence data and degenerate primer polymerase chain reaction to clone two full-length human P5CDh cDNAs, differing in length by 1 kilobase pair (kb). Both cDNAs have the identical 1689-base pair open reading frame encoding a protein of 563 residues with a predicted molecular mass of 62 kDa. The long cDNA contains an additional 1-kb insert in the 3'-untranslated region that appears to be an alternatively spliced intron. The conceptual translation of human P5CDh has 89% sequence identity with the published human P5CDh peptide sequences and 42 and 26% identity with Saccharomyces cerevisiae and Escherichia coli P5CDhs, respectively, as well as homology to several other aldehyde dehydrogenases. Both P5CDh cDNA clones detect a single 3.2-kb transcript on Northern blots of multiple human tissues, indicating the long cDNA containing the 3'-untranslated intron represents the predominant transcript. The P5CDh structural gene appears to be single copy with a size of about 20 kb localized to chromosome 1. To confirm the identity of the putative P5CDh cDNAs, we expressed them in a P5CDh-deficient strain of S. cerevisiae. Both conferred measurable P5CDh activity and the ability to grow on proline as a sole nitrogen source.
Asunto(s)
Escherichia coli/enzimología , Pirrolina Carboxilato Reductasas/biosíntesis , Saccharomyces cerevisiae/enzimología , 1-Pirrolina-5-Carboxilato Deshidrogenasa , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Femenino , Biblioteca de Genes , Humanos , Masculino , Datos de Secuencia Molecular , Peso Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/genética , Ratas , Proteínas Recombinantes , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
Graves' hyperthyroidism is due primarily to overproduction of antibodies to thyrotropin receptors (TR-ab), which stimulate the thyroid gland and cause hyperthyroidism. Antibody production during antithyroid drug therapy is an important determinant of the course of the disease. We therefore observed the changes of serum TR-ab, thyroglobulin (Tg) and thyroid hormone levels in response to administration of L-thyroxine (T4) in Graves' hyperthyroid patients during antithyroid drug therapy. Serum levels of TR-ab, Tg and other thyroid hormones were measured by radioimmunoassay (RIA) during either methimazole treatment alone or in combination with thyroxine in 60 Graves' hyperthyroid patients. The patients initially were treated with 30 mg of methimozole daily for 3 months, which was then reduced to 15 mg daily for the following 3 months. All patients were euthyroid 6 months after the start of antithyroid therapy and the TR-ab level decreased from 61 +/- 11% (+/- SD) to 28 +/- 7% (p < 0.01). Patients then were divided into three groups: group A (N = 25), whose TR-ab level was 10% or more (the cut-off value for positivity), received 0.1 mg of T4 and 10 mg of methimazole daily for 6 months; group B (N = 15), whose TR-ab level also was 10% or more and was age- and thyroid function-matched with group A, received only 10 mg of methimazole daily for 6 months; group C (N = 20), with a TR-ab level of less than 10%, received 10 mg of methimazole alone daily for 6 months.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos/análisis , Enfermedad de Graves/sangre , Enfermedad de Graves/tratamiento farmacológico , Metimazol/uso terapéutico , Receptores de Tirotropina/inmunología , Receptores de Tirotropina/metabolismo , Tiroglobulina/sangre , Tiroxina/uso terapéutico , Adolescente , Adulto , Quimioterapia Combinada , Femenino , Enfermedad de Graves/patología , Humanos , Masculino , Persona de Mediana Edad , Glándula Tiroides/patología , Hormonas Tiroideas/sangreRESUMEN
Proline prototrophy was restored to an Escherichia coli proBA proline auxotroph by ornithine and a mothbean (Vigna aconitifolia) cDNA expression library. This novel strategy, "trans-complementation," allowed isolation of a cDNA encoding ornithine delta-aminotransferase (delta-OAT). This enzyme transaminates ornithine to glutamic-gamma-semialdehyde (GSA), thereby bypassing the block in GSA synthesis from glutamate in the proBA mutant. The identity of the mothbean enzyme was confirmed by its high sequence homology to mammalian and yeast delta-OATs as well as to a family of bacterial and fungal omega-aminotransferases and an absence of significant homology to various alpha-aminotransferases. The V. aconitifolia OAT cDNA encodes a polypeptide of 48.1 kDa. The native enzyme expressed in E. coli appears to be a monomer with Km of 2 mM for ornithine and 0.75 mM for alpha-ketoglutarate. Levels of mRNA in V. aconitifolia for delta 1-pyrroline-5-carboxylate synthetase (P5CS) and delta-OAT, the two key enzymes for proline synthesis, were monitored under different physiological conditions. Salt stress and nitrogen starvation induced P5CS mRNA levels and depressed OAT mRNA levels. Conversely, OAT mRNA level was elevated in plants supplied with excess nitrogen while the P5CS mRNA level was reduced. These data suggest that the glutamate pathway is the primary route for proline synthesis in plants during conditions of osmotic stress and nitrogen limitation whereas the ornithine pathway assumes prominence under high nitrogen input.
Asunto(s)
Clonación Molecular , ADN/genética , Escherichia coli/enzimología , Ornitina-Oxo-Ácido Transaminasa/genética , Plantas/enzimología , Prolina/biosíntesis , 1-Pirrolina-5-Carboxilato Deshidrogenasa , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Fabaceae , Glutamatos/metabolismo , Ácido Glutámico , Cinética , Datos de Secuencia Molecular , Mutación , Nitrógeno/administración & dosificación , Nitrógeno/farmacología , Ornitina-Oxo-Ácido Transaminasa/química , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Presión Osmótica , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Plantas/efectos de los fármacos , Plantas/genética , Plantas Medicinales , ARN Mensajero/metabolismo , Homología de SecuenciaRESUMEN
Patients with nontoxic thyroid nodules are often treated with thyroxine (T4) in order to reduce the size of the nodule, but the efficacy of thyrotropin-suppressive therapy with thyroxine remains uncertain. In this study, 35 patients with a solitary thyroid nodule were given thyroxine (0.1 mg/day) for three months. High resolution (7.5 MHz) sonography was used to measure the size of the nodules before and after thyroxine therapy. The volume of the nodules in 12 patients (34.3%, responders) decreased by more than 50% after thyroxine therapy. In this group, the mean serum thyroglobulin (Tg) level decreased significantly (from 424 to 107 ng/mL, p < 0.001). In the nonresponders, the mean serum Tg level did not change significantly after thyroxine therapy (291 vs 261 ng/mL, p > 0.05). The mean serum total T4 and free T4 concentrations increased significantly in both groups after thyroxine therapy (p < 0.01), the serum total triiodothyronine (T3) level did not change (p > 0.05), and the serum thyrotropin level (TSH) and T3/T4 ratio decreased markedly (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tiroglobulina/sangre , Nódulo Tiroideo/tratamiento farmacológico , Tiroxina/uso terapéutico , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nódulo Tiroideo/sangre , Nódulo Tiroideo/diagnóstico por imagen , Tirotropina/sangre , Tiroxina/sangre , Triyodotironina/sangre , UltrasonografíaRESUMEN
Many plants synthesize and accumulate proline in response to osmotic stress. Despite the importance of this pathway, however, the exact metabolic route and enzymes involved in the synthesis of proline in plants have not been unequivocally identified. We report here the isolation of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in proline biosynthesis. The two enzymatic domains of P5CS correspond to the ProB and ProA proteins of Escherichia coli and contain a leucine zipper in each domain, which may facilitate inter- or intramolecular interaction of this protein. The Vigna P5CS enzyme activity is feedback regulated by proline but is less sensitive to end-product inhibition than is the E. coli gamma-glutamyl kinase. The P5CS gene is expressed at high levels in Vigna leaves and is inducible in roots subjected to salt stress, suggesting that P5CS plays a key role in proline biosynthesis, leading to osmoregulation in plants.
Asunto(s)
Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Fosfotransferasas (aceptor de Grupo Carboxilo) , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Plantas/enzimología , Prolina/biosíntesis , 1-Pirrolina-5-Carboxilato Deshidrogenasa , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN/genética , ADN/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Retroalimentación , Glutamato-5-Semialdehído Deshidrogenasa , Cinética , Datos de Secuencia Molecular , Plantas/genética , Poli A/genética , Poli A/aislamiento & purificación , Prolina/farmacología , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero , Homología de Secuencia de AminoácidoRESUMEN
A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.